CN104105708A

CN104105708A - Pdgf receptor beta binding polypeptides

Info

Publication number: CN104105708A
Application number: CN201280068404.3A
Authority: CN
Inventors: 陈岩; R·W·瓦格纳; C·帕兹马尼
Original assignee: BODY BIOSCIENCES X
Current assignee: BODY BIOSCIENCES X; X Body Inc
Priority date: 2011-12-05
Filing date: 2012-12-05
Publication date: 2014-10-15
Anticipated expiration: 2032-12-05
Also published as: JP6843816B2; AU2012347972A1; TWI591074B; EP2788379A4; EP2788379B1; CA2857721C; US20200299390A1; TWI640537B; EP3712173B1; CN107353342A; US20220089748A1; JP6483442B2; US20130177572A1; AU2012347972B2; CN104105708B; US9416179B2; JP2015501814A; US11136398B2; US20170029510A1; EP2788379A1

Abstract

The present invention provides binding polypeptides (e.g., antibodies or fragments thereof) that specifically bind to a target antigen (e.g., a human antigen, e.g., human PDGFRbeta) with high affinity. The invention also provides, libraries of binding polypeptides, pharmaceutical compositions, as well as nucleic acids encoding binding polypeptides, recombinant expression vectors and host cells for making such binding polypeptides. Methods of using binding polypeptide of the invention to diagnose and treat disease are also encompassed by the invention.

Description

Pdgf receptor β Binding peptide

The application advocates the right of priority of the U.S. Provisional Application 61/610,905 of the U.S. Provisional Application application on March 14th, 61/566,778 and 2012 of application on December 5th, 2011, and the mode that described provisional application is all quoted is in full incorporated herein.

Background technology

Platelet-derived growth factor (PDGF) is powerful mitogen and the chemoattractant in the movable cell of originating of a leaf and the pathology that relate to numerous disease.The dipolymer that PDGF connects with disulphide exists, and described dipolymer is made up of two homology chain A or B that form three kinds of unique PDGF isotype AA, BB or AB capable of being combined.All isotypes of PDGF all mediate its mitogenesis effect by being bonded to cell surface pdgf receptor (PDGFR).

Pdgf receptor belongs to family tyrosine kinase and is made up of two kinds of acceptor isotype α and β.α and β isotype can be distinguished by its unique ligand binding specificity.PDGF beta receptor can only be bonded to B chain (isotype BB and AB), and PDGF α acceptor can be bonded to all isotypes of PDGF.

The combination of PDGF and cell surface PDGFR causes receptor dimerization and trans autophosphorylation, and then causes again Cellular Signaling Transduction Mediated event, and described event especially causes cell proliferation and cell migration.Therefore, the PDGFR antagonist of blocking-up PDGF combination and/or receptor dimerization can activate relevant disease to PDGFR in order to treatment or prevention.

Therefore, needing in the art can be in order to treat the novel PDGFR antagonist that activates relevant disease to PDGFR.

Summary of the invention

For example the invention provides with high-affinity specific binding, for example, for example, in the Binding peptide (antibody or its fragment) of target antigen (mankind's antigen, mankind PDGF).In a preferred embodiment, the invention provides the Binding peptide that is incorporated into PDGFR β (for example mankind PDGFR β) and antagonism PDGFR β activation with high-affinity.Such Binding peptide is particularly useful for treating PDGFR ss related diseases or illness (macular degeneration (AMD) that for example age is relevant).The present invention also provides library, the pharmaceutical composition of Binding peptide, and nucleic acid, recombinant expression vector and the host cell for the manufacture of such Binding peptide of coding Binding peptide.The method that detects PDGFR β and adjusting PDGFR 'beta ' activity with Binding peptide of the present invention is also contained in the present invention.

Therefore, on the one hand, the invention provides through separation and combination polypeptide, it comprises VH territory, and wherein as separate domains, VH territory is incorporated into antigen with the Kd that is less than 100pM.

On the other hand, the invention provides specific binding in PDGFR β through separation and combination polypeptide, it comprises the CDR3 sequence as shown in SEQ ID NO:1.

In certain embodiments, Binding peptide comprises VH territory, and described VH territory comprises the HCDR3 aminoacid sequence shown in SEQ ID NO:1.VH territory can further comprise the HCDR2 that comprises the aminoacid sequence that is selected from SEQ ID NO:2-32 and/or the HCDR1 that comprises the aminoacid sequence that is selected from SEQ ID NO:33-62.

In certain embodiments, polypeptide comprises VH territory, and described VH territory comprises with the VH domain amino acid sequence that is selected from SEQ ID NO:318-368 and enjoy at least 80% the aminoacid sequence of (for example 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) amino acid identity.

In certain embodiments, polypeptide comprises VH territory, and described VH territory comprises the aminoacid sequence that is selected from SEQ ID NO:318-368.

In certain embodiments, Binding peptide comprises VL territory.VH territory can further comprise the LCDR3 that comprises the aminoacid sequence that is selected from SEQ ID NO:63-147, the LCDR2 that comprises the aminoacid sequence that is selected from SEQ ID NO:148-232 and/or the LCDR1 that comprises the aminoacid sequence that is selected from SEQ ID NO:233-317.

In certain embodiments, polypeptide comprises VL territory, and described VL territory comprises with the VL domain amino acid sequence that is selected from SEQ ID NO:369-453 and enjoy at least 80% the aminoacid sequence of (for example 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) amino acid identity.

In certain embodiments, polypeptide comprises VL territory, and described VL territory comprises the aminoacid sequence that is selected from SEQ ID NO:369-453.

In other side, the invention provides Binding peptide, it is incorporated into the identical epi-position on PDGFR β with the Binding peptide that comprises the VH domain amino acid sequence shown in SEQ ID No:318.In a preferred embodiment, Binding peptide comprises the VH domain amino acid sequence of enjoying at least 80% amino acid identity (for example 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) with the VH domain amino acid sequence that is selected from SEQ ID NO:318-368.

On the other hand, the invention provides Binding peptide, it is incorporated into PDGFR β with the Binding peptide competition that comprises the VH domain amino acid sequence as shown in SEQ ID No:318.In a preferred embodiment, Binding peptide comprises the VH domain amino acid sequence of enjoying at least 80% amino acid identity with the VH domain amino acid sequence that is selected from SEQ ID NO:318-368.

In certain embodiments, Binding peptide of the present invention suppresses PDGFR 'beta ' activity.In one embodiment, PDGFR 'beta ' activity suppresses by the combination of antagonism PDGF and PDGFR β.In another embodiment, PDGFR 'beta ' activity suppresses by antagonism PDGFR β dimerization.

In certain embodiments, Binding peptide of the present invention is to be less than the Kd of 100pM and/or to be less than 10 ^-3s ^-1dissociation rate be incorporated into PDGFR β.

In certain embodiments, Binding peptide specific binding of the present invention is in mouse and mankind PDGFR β.

In certain embodiments, Binding peptide of the present invention to be to be less than the IC50 antagonism PDGF of 5nM and the combination of PDGFR β, to suppress the part inducibility tyrosine phosphorylation of PDGFR β, suppress the migration of retina pericyte to be less than the IC50 of 6nM to be less than the IC50 of 4nM, and/or has the melt temperature (Tm) of at least 68 DEG C.

On the other hand, the invention provides coding Binding peptide of the present invention through isolating nucleic acid.

On the other hand, the invention provides the recombinant expression vector through isolating nucleic acid that comprises the Binding peptide of the present invention of encoding.

On the other hand, the invention provides the host cell of expressing Binding peptide of the present invention.

On the other hand, the invention provides and manufacture specific binding in the method for the Binding peptide of mankind PDGFR β, it comprises that the host cell that can express Binding peptide of the present invention cultivates described host cell is produced under the condition of Binding peptide.

On the other hand, the invention provides pharmaceutical composition, it comprises Binding peptide of the present invention and one or more of pharmaceutically acceptable carrier (carrier).

On the other hand, the invention provides the method that is used for the treatment of disease or illness PDGFR ss related diseases or illness (macular degeneration (AMD) or cancer that for example age is relevant), described method comprises to the experimenter who has needs uses pharmaceutical composition of the present invention.

On the other hand, the invention provides not various library in VH territory in pairs, the each member of its Chinese library is incorporated into mankind PDGFR β.In a preferred embodiment, the each member in library comprises the CDR3 aminoacid sequence shown in SEQ ID NO:1 and is the diversity in FR1-FR3 region.In a preferred embodiment, library is nucleic acid display libraries (for example DNA display libraries).

On the other hand, the invention provides and stablize various library that VH/VL is right, the each member of its Chinese library is incorporated into mankind PDGFR β.In a preferred embodiment, the each member in library comprises VH territory, and described VH territory comprises the CDR3 aminoacid sequence shown in SEQ ID NO:1.In a preferred embodiment, VL territory is mankind VL territory.In a preferred embodiment, library is nucleic acid display libraries (for example DNA display libraries).

Brief description of the drawings

Fig. 1 is the schematic diagram building for the exemplary VH territory nucleic acid display libraries of disclosed method.

Fig. 2 describes to measure XB1511VH territory, unselected XB1511CDR3/ framework reorganizes DNA display libraries (R0) and XB1511CDR3/ framework reorganization DNA display libraries pond is rear in four-wheel selection (R4) and the external binding analysis result of the combination of the mankind or mouse PDGFR β.

Fig. 3 describes the result of the surface plasma body resonant vibration binding of the binding kinetics that measures XB1511 and framework reorganization derivative XB2202 and XB2708 and mankind PDGFR β.

Fig. 4 describes the result of the surface plasma body resonant vibration binding analysis of the binding kinetics that measures XB2202 and the mankind (A) and mouse (B) PDGFR β.

Fig. 5 describes the result of the surface plasma body resonant vibration binding analysis of the binding kinetics that measures XB2708 and the mankind (A) and mouse (B) PDGFR β.

Fig. 6 is the schematic diagram for the structure of the exemplary VL nucleic acid display libraries of disclosed method.

Fig. 7 describes to measure to comprise from second of the paired DNA displaying of VH/VL screening and takes turns the result of screening the XB1511/VL scFv in VL territory of pond separation and the elisa assay of the combination of mankind PDGFR β.

Fig. 8 describes to measure the result comprising from the XB1511/VL scFv in VL territory of third round screening pond separation and the elisa assay of the combination of mankind PDGFR β of the paired DNA displaying of VH/VL screening.

Fig. 9 describes to measure to comprise from second of the paired DNA displaying of VH/VL screening and takes turns the result of screening the XB2202/VL scFv in VL territory of pond separation and the elisa assay of the combination of mankind PDGFR β.

Figure 10 describes to measure the result from the elisa assay of the not paired VL territory of the XB1511/VL scFv shown in Fig. 9 and the combination of mankind PDGFR β.

Figure 11 describes to measure ³⁵the XB1511VH territory of S Met mark and the result of showing the research of the solution binding affinity containing the scFV of XB1511 and the combination of mankind PDGFR β of screening available from the paired DNA of VH/VL.

Figure 12 describes to measure ³⁵the XB2202VH territory of S Met mark and the result of showing the research of the solution binding affinity containing the scFV of XB2202 and the combination of mankind PDGFR β of screening available from the paired DNA of VH/VL.

Figure 13 describes to measure the result of dynamic (dynamical) surface plasma body resonant vibration competitive binding assay that PDGF-BB is combined with PDGFR β under the XB2202 of various concentration.

Figure 14 describes the result of the cell in vitro migration analysis that measures the inhibition of XB2708 to pericyte migration.

Figure 15 describes to measure the result that suppresses the unmarked migration analysis of human foreskin fiber's parent cell transfer ability containing the IgG1 of XB1511.

Figure 16 describes to measure XB2202VH territory and XB2202/A4scFv is hatched afterwards and the result of the elisa assay of the combination of mankind PDGFR β at various temperatures.

Embodiment

For example the invention provides with high-affinity specific binding, for example, in the Binding peptide (antibody or its fragment) of target antigen (mankind's antigen).In a preferred embodiment, Binding peptide of the present invention is incorporated into PDGFR β (for example mankind PDGFR β) and suppresses PDGFR 'beta ' activity with high-affinity.Such Binding peptide is particularly useful for treating PDGFR ss related diseases or illness (macular degeneration (AMD) that for example age is relevant).The present invention also provides nucleic acid, recombinant expression vector and the host cell for the manufacture of such Binding peptide of library, pharmaceutical composition and the coding Binding peptide of Binding peptide.The method that detects PDGFR β and adjusting PDGFR 'beta ' activity with Binding peptide of the present invention is also contained in the present invention.

I: definition

For the present invention can be understood relatively easily, first define some term.

As used herein, term " PDGFR β " refers to platelet-derived growth factor receptor β.PDGFR beta nucleoside well known in the art acid and peptide sequence.Exemplary mankind PDGFR β amino sequence is set forth in GenBank preservation GI:4505683, and exemplary mouse PDGFR β amino sequence is set forth in GenBank preservation GI:226371752.

As used herein, term " PDGF " refers to platelet-derived growth factor.PDGF Nucleotide well known in the art and peptide sequence.Exemplary mankind PDGF amino sequence is set forth in GenBank preservation GI:4505681, and exemplary mouse PDGF amino sequence is set forth in GenBank preservation GI:400744.

As used herein, term " Binding peptide " refers to that the antigen binding site that contains antibody all or part of (all or part of of for example heavy chain and/or light chain variable territory, at least HCDR3 of for example heavy chain variable domain) is so that the polypeptide of Binding peptide specific recognition target antigen.The limiting examples of Binding peptide comprises antibody or its fragment, and has become all or part of immunoglobulin-like territory (for example fibronectin territory) of the antigen binding site that comprises antibody.

As used herein, term " antibody " refers to comprise four polypeptide chains, two heavy chain (H) chains that interconnect by cystine linkage and the immunoglobulin molecules of two light (L) chains, with and polymer (for example IgM).Each heavy chain comprises variable region of heavy chain (being abbreviated as VH) and CH.CH comprises three territories, i.e. CH1, CH2 and CH3.Each light chain comprises variable region of light chain (being abbreviated as VL) and constant region of light chain.Constant region of light chain comprises a territory (CL1).VHJi VL district can further be subdivided into the hypervariable region that is called complementary determining region (CDR), is interspersed with the more conserved regions that is called framework region (FR).

As used herein, " antigen-binding portion thereof " of term antibody comprises that specific binding antigen is to form any naturally occurring with obtainable synthetic or genetically engineered polypeptide or the glucoprotein of enzymatic mode of mixture.The Fab of antibody can use any applicable standard technique for example to come from complete antibody molecule, described standard technique is such as being proteolytic digestion or genetic recombination through engineering approaches technology, and it relates to operation and the variable and optional constant domain of expressible dna encoding antibody.The limiting examples of antigen-binding portion thereof comprises: (i) Fab fragment; (ii) F (ab') 2 fragments; (iii) Fd fragment; (iv) Fv fragment; (v) scFv (scFv) molecule; (vi) dAb fragment; And (vii) the minimum recognition unit that for example, formed by the amino-acid residue of analog antibody hypervariable region (through separate complementary determining region (CDR)).The molecule of other through engineering approaches is also contained in statement " antigen-binding portion thereof ", such as double antibody (diabody), three antibody (triabody), four antibody (tetrabody) and miniantibody.

As used herein, term " VH territory " and " VL territory " refer to respectively antibody single variable heavy chain and light chain territory, it comprises FR (framework region) 1,2,3 and 4 and CDR (complementary determining region) 1,2 and 3 (referring to people such as Kabat, (1991) Sequences of Proteins of Immunological Interest. (NIH publishes the 91-3242 phase, Bethesda).

As used herein, term " FR1-FR3 " refers to contain FR1, CDR2, FR2, CDR2 and FR3 but gets rid of VH district of CDR3 JiFR4 district.

As used herein, term " not paired " refers to that VH or VL do not connect respectively (covalently or non-covalently) in complementary VL or VH territory.

As used herein, term " complementary VL or VH territory " refers to be connected to form with VH or VL territory VL or the VH territory that VH/VL is right respectively.

As used herein, term " VH/VL to " refers to the non-covalent dimer in single VH and single VL territory, wherein VL and VH territory are connected to be similar to viewed mode in complete four poly-immunoglobulin molecules, and described dimer can specific binding at least one target antigen." stablize VH/VL to " be not for showing the VH/VL couple significantly dissociating in substituting group VH and VL territory under physiological condition.

As used herein, term " CDR " or " complementary determining region " meaning is the discrete antigen binding site seeing in the variable region of heavy chain and light chain polypeptide.The people such as Kabat, J.Biol.Chem.252, the people such as 6609-6616 (1977) and Kabat, Sequences of protein of immunological interest. (1991), and the people such as Chothia, the people such as J.Mol.Biol.196:901-917 (1987) and MacCallum, J.Mol.Biol.262:732-745 (1996) has described these specific regions, and wherein said definition comprises overlapping or the subset of amino-acid residue when time compared to each other.Elaboration contain as above-cited reference separately the amino-acid residue of defined CDR so that comparison.Preferably, term " CDR " is if Kabat is based on the more defined CDR of sequence.

As used herein, term " framework (FR) amino-acid residue " refers to those amino acid in the framework region of immunoglobulin chain.As used herein, term " framework region " or " FR district " comprise as the part of variable region but not the amino-acid residue of the part of CDR (for example, using the CDR of Kabat definition).

As used herein, term " specific binding in " refers to that Binding peptide is with at least about 1 × 10 ^-6m, 1 × 10 ^-7m, 1 × 10 ^-8m, 1 × 10 ^-9m, 1 × 10 ^-10m, 1 × 10 ^-11m, 1 × 10 ^-12m or more Kd are incorporated into antigen, and/or affinity of heterogenetic antigen affinity that at least twice is large is incorporated into than it ability of antigen.But should be appreciated that, Binding peptide can specific binding in two or more antigen relevant in sequence.For example, Binding peptide of the present invention can specific binding for example, in the mankind and both PDGFR β of non-human (mouse or non-human primate).

As used herein, term " antigen " refers to binding site or the epi-position identified by Binding peptide.

As used herein, term " nucleic acid display libraries " refers to the displaying that any technical generally acknowledged external acellular phenotype-genotype connects, and it is including but not limited to for example shown below: United States Patent (USP) the 7th, 195, No. 880; The 6th, 951, No. 725; The 7th, 078, No. 197; The 7th, 022, No. 479; The 6th, 518, No. 018; The 7th, 125, No. 669; The 6th, 846, No. 655; The 6th, 281, No. 344; The 6th, 207, No. 446; The 6th, 214, No. 553; The 6th, 258, No. 558; The 6th, 261, No. 804; The 6th, 429, No. 300; The 6th, 489, No. 116; The 6th, 436, No. 665; The 6th, 537, No. 749; The 6th, 602, No. 685; The 6th, 623, No. 926; The 6th, 416, No. 950; The 6th, 660, No. 473; The 6th, 312, No. 927; The 5th, 922, No. 545; And the 6th, 348, No. 315, and WO2010/011944, the mode that described patent is all quoted is in full incorporated herein.

As used herein, term " carrier " is intended to refer to carry the nucleic acid molecule of its another nucleic acid having connected.The carrier of one type is " plasmid ", and it refers to connect the circular double-stranded DNA ring of other DNA section.The carrier of another type is virus vector, and wherein other DNA section can be connected to viral genome.For example, in host cell that some carrier can be introduced at it (, there is the bacteria carrier of bacterium replication orgin, and sequestered Mammals carrier) autoduplication.Other carrier (for example non-sequestered Mammals carrier) can be integrated in the genome of host cell in the time introducing in host cell, copies thus together with host genome.In addition, some carrier can instruct the expression of its gene being operably connected to.Such carrier is called " recombinant expression vector " (or abbreviation " expression vector ") in this article.Generally speaking, the effectiveness of expression vector in recombinant DNA technology is often plasmid form.Term " plasmid " is used interchangeably with " carrier ".But, this invention is intended to comprise other form of such expression vector, for example, such as virus vector (, replication defective retrovirus, adenovirus and adeno-associated virus), it provides equivalent function.

As used herein, term " host cell " is intended to refer to introduce the cell of recombinant expression vector.Should be appreciated that, this term is not only intended to refer to specific subject cell, and refers to the filial generation of this like cell.Because may be because some amendment appears in sudden change or environmental influence in offspring, so these filial generation actual capabilities are not identical with parental cell, but still be included in the category of term " host cell " as used herein.

As used herein, term " treatment (treat) ", " (treating) for the treatment of ", " treatment (treatment) " refer to treatment as herein described or preventive measures." treatment " method adopts to experimenter, the experimenter who for example suffers from PDGFR ss related diseases or illness (for example AMD) or easily suffer from this disease or illness uses antibody of the present invention or antigen-binding portion thereof, with prevention, healing, delay disease or illness or recurrent disease or illness, its seriousness that reduces, or improve its one or more of symptoms, or to extend the expection survival time of experimenter's survival time while treating without this to exceed.

As used herein, term " PDGFR ss related diseases or illness " comprises the disease patient's condition and/or the symptom relevant to PDGFR 'beta ' activity.Exemplary PDGFR ss related diseases or illness include, but is not limited to relevant macular degeneration of age (AMD) and cancer.

As used herein, term " significant quantity " refers to when the amount of Binding peptide for the treatment of, prognosis or diagnosis that is enough to realize PDGFR ss related diseases as described herein or illness in the time that experimenter uses.Treatment significant quantity changes seriousness, the method for application etc. of the experimenter depending on treated and disease symptom, experimenter's body weight and age, disease symptom, and it can be determined easily by those of ordinary skill in the art.Application dosage can be between the approximately 1ng of for example Binding peptide of the present invention to approximately 10, and 000mg, approximately 1 μ g are to approximately 5, and 000mg, about 1mg are to approximately 1, and 000mg, about 10mg are to about 100mg.Capable of regulating dosage regimen is to provide optimum therapeutic response.Significant quantity is also for any toxicity or the deleterious effect (that is side effect) of Binding peptide have all been reduced to the prior amount of minimum and/or advantageous effects.

As used herein, term " experimenter " comprises any mankind or non-human animal.

As used herein, term " surface plasma body resonant vibration " refers to optical phenomena, and it allows by for example using BIAcore ^tMsystem (Biacore Life Sciences division of GE Healthcare, Piscataway, NJ) is carried out the intramatrical protein concn of detection of biological sensor and is changed to analyze real-time interaction.

As used herein, term " K _d" refer to the equilibrium dissociation constant of particular combination polypeptide/AI.

As used herein, term " dissociation rate " refers to the dissociation rate (K of particular combination polypeptide/AI _off).

As used herein, term " epi-position " refers in binding molecule of the present invention and the interactional antigenic determinant of specific antigens binding site.Single antigen can have more than one epi-position.Therefore, different antibodies can be incorporated into the different zones on antigen and can have different biological actions.Epi-position can be conformation or linear.Conformational epitope produces by the space amino acid arranged side by side of the different sections from linear polypeptide chain.Linear epitope is the epi-position producing by adjacent amino acid residue in polypeptide chain.

II. Binding peptide

On the one hand, the invention provides the Binding peptide that comprises VH territory, wherein as through separate domains, VH territory for example, is incorporated into antigen with the Kd that is less than about 200pM (approximately 200,190,180,175,170,160,150,140,130,120,110,100,95,90,80,75,70,65,60,55,50,40,30,20,10,5 or 1pM or still less).

On the other hand, for example the invention provides specific binding, in the Binding peptide (, antibody or its Fab) of PDGFR β and inhibition PDGFR 'beta ' activity.Such Binding peptide is particularly useful for treating PDGFR ss related diseases or illness (macular degeneration that for example age is relevant or AMD).

Generally speaking, PDGFR β Binding peptide of the present invention comprises specific binding in the heavy chain CDR3 of PDGFR β (HCDR3) aminoacid sequence.A kind of non-limiting HCDR3 sequence that is applicable to Binding peptide of the present invention is the heavy chain CDR3 aminoacid sequence as shown in SEQ ID NO:1 (HGGDRSY).In other embodiments, heavy chain CDR3 sequence is the variant of SEQ ID NO:1, and it comprises at least one (for example one, two or three) conserved amino acid with respect to SEQ ID NO:1 and replaces.

Can merge specific binding for example, all can be used in Binding peptide of the present invention in any Binding peptide of the heavy chain CDR3 of PDGFR β aminoacid sequence (the CDR3 aminoacid sequence shown in SEQ ID NO:1 (HGGDRSY)), be including but not limited to antibody or its fragment, and immunoglobulin-like territory.Applicable immunoglobulin-like territory is including but not limited to fibronectin territory (referring to people such as such as Koide, (2007), Methods Mol.Biol.352:95-109, its mode of quoting is in full incorporated herein), DARPin is (referring to people such as such as Stumpp, (2008) Drug Discov.Today 13 (15-16): 695-701, its mode of quoting is in full incorporated herein), the Z territory of a-protein is (referring to people such as Nygren, (2008) J.275 (11): 2668-76 of FEBS, its mode of quoting is in full incorporated herein), lipocalin protein (Lipocalin) (referring to, the people such as such as Skerra, (2008) J.275 (11): 2677-83 of FEBS, its mode of quoting is in full incorporated herein), Affilin (without translation in correspondence) is (referring to people such as such as Ebersbach, (2007) J.Mol.Biol.372 (1): 172-85, its mode of quoting is in full incorporated herein), Affitin is (referring to people such as such as Krehenbrink, (2008) .J.Mol.Biol.383 (5): 1058-68, its mode of quoting is in full incorporated herein), Avimer (without translation in correspondence) is (referring to people such as such as Silverman, (2005) Nat.Biotechnol.23 (12): 1556-61, its mode of quoting is in full incorporated herein), Fynomer (without translation in correspondence) is (referring to people such as such as Grabulovski, (2007) J Biol Chem 282 (5): 3196-3204, its mode of quoting is in full incorporated herein) and Ku Nizi (Kunitz) territory peptide (referring to, the people such as such as Nixon, (2006) Curr Opin Drug Discov Devel 9 (2): 261-8, its mode of quoting is in full incorporated herein).

In a preferred embodiment, PDGFR β Binding peptide is antibody or its Fab, and it comprises VH territory and/or VL territory.In table 1-4, set forth and be applicable to exemplary CDR of the present invention, VH and VL aminoacid sequence.Therefore, in certain embodiments, Binding peptide can comprise HCDR3 (SEQ ID NO:1) and HCDR2 and/or HCDR1 sequence, its independently selected from the heavy chain HCDR2 shown in table 1 or HCDR1 sequence any one.In certain embodiments, Binding peptide of the present invention can further comprise light chain CDR, its independently selected from light chain CDR1, the CDR2 shown in table 2 or CDR3 sequence any one.For example, Binding peptide of the present invention can comprise any one in the weight chain variable shown in table 3 (VH) territory, optionally with the light chain variable shown in table 4 (VL) territory in any one pairing.

Table 1: the heavy chain cdr amino acid sequence of exemplary anti-PDGFR β antibody

Table 2: the light chain cdr amino acid sequence of exemplary anti-PDGFR β antibody

Table 3: heavy chain variable domain (VH) aminoacid sequence of exemplary anti-PDGFR β antibody

Table 4: light chain variable territory (VL) aminoacid sequence of exemplary anti-PDGFR β antibody

In certain embodiments, the heavy chain CDR3 sequence that antibody or its Fab comprise SEQ ID NO:1 and one or more are selected from the CDR region amino acid sequence of SEQ ID NO:2-317.In exemplary embodiment, antibody or its Fab comprise and are selected from respectively following HCDR3, HCDR2 and HCDR1 aminoacid sequence: SEQ ID NO:1,2 and 3; 1,2 and 34; 1,3 and 35; 1,4 and 35; 1,5 and 36; 1,6 and 36; 1,3 and 36; 1,7 and 36; 1,8 and 36; 1,9 and 36; 1,10 and 38; 1,2 and 38; 1,11 and 39; 1,12 and 40; 1,13 and 41; 1,13 and 42; 1,13 and 33; 1,14 and 43; 1,14 and 44; 1,15 and 45; 1,16 and 45; 1,17 and 46; 1,18 and 47; 1,19 and 47; 1,20 and 48; 1,2 and 49; 1,21 and 50; 1,2 and 51; 1,2 and 52; 1,2 and 53; 1,22 and 54; 1,23 and 55; 1,24 and 56; 1,25 and 46; 1,26 and 57; 1,27 and 58; 1,28 and 59; 1,29 and 60; 1,30 and 61; 1,31 and 62; And 1,32 and 62.

In other embodiments, antibody or its Fab further comprise and are selected from respectively following LCDR3, LCDR2 and LCDR1 aminoacid sequence: SEQ ID NO:63,148 and 233; 64,149 and 234; 65,150 and 235; 66,151 and 236; 67,152 and 237; 68,153 and 238; 69,154 and 239; 70,155 and 240; 71,156 and 241; 72,157 and 242; 73,158 and 243; 741,159 and 244; 75,160 and 245; 76,161 and 246; 77,162 and 247; 78,163 and 248; 79,164 and 249; 80,165 and 250; 81,166 and 251; 82,167 and 252; 83,168 and 253; 84,169 and 254; 85,170 and 255; 86,171 and 256; 87,172 and 257; 88,173 and 258; 89,174 and 259; 90,175 and 260; 91,176 and 261; 92,177 and 262; 93,178 and 263; 94,179 and 264; 95,180 and 265; 96,181 and 266; 97,182 and 267; 98,183 and 268; 99,184 and 269; 100,185 and 270; 101,186 and 271; 102,187 and 272; 103,188 and 273; 104,189 and 274; 105,190 and 275; 106,191 and 276; 107,192 and 277; 108,193 and 278; 109,194 and 279; 110,195 and 280; 111,196 and 281; 112,197 and 282; 113,198 and 283; 114,199 and 284; 115,200 and 285; 116,201 and 286; 117,202 and 287; 118,203 and 288; 119,204 and 289; 120,205 and 290; 121,206 and 291; 122,207 and 292; 123,208 and 293; 124,209 and 294; 125,210 and 295; 126,211 and 296; 127,212 and 297; 128,213 and 298; 129,214 and 299; 130,215 and 300; 131,216 and 301; 132,217 and 302; 133,218 and 303; 134,219 and 304; 135,220 and 305; 136,221 and 306; 137,222 and 307; 138,223 and 308; 139,224 and 309; 140,225 and 310; 141,226 and 311; 142,227 and 312; 143,228 and 313; 144,229 and 314; 145,220 and 315; 146,231 and 316; And 147,232 and 317.

In other embodiments, antibody or its Fab comprise shown in SEQ ID NO:1 HCDR3 aminoacid sequence, and be selected from respectively following LCDR3, LCDR2 and LCDR1 aminoacid sequence: SEQ ID NO:63,148 and 233; 64,149 and 234; 65,150 and 235; 66,151 and 236; 67,152 and 237; 68,153 and 238; 69,154 and 239; 70,155 and 240; 71,156 and 241; 72,157 and 242; 73,158 and 243; 741,159 and 244; 75,160 and 245; 76,161 and 246; 77,162 and 247; 78,163 and 248; 79,164 and 249; 80,165 and 250; 81,166 and 251; 82,167 and 252; 83,168 and 253; 84,169 and 254; 85,170 and 255; 86,171 and 256; 87,172 and 257; 88,173 and 258; 89,174 and 259; 90,175 and 260; 91,176 and 261; 92,177 and 262; 93,178 and 263; 94,179 and 264; 95,180 and 265; 96,181 and 266; 97,182 and 267; 98,183 and 268; 99,184 and 269; 100,185 and 270; 101,186 and 271; 102,187 and 272; 103,188 and 273; 104,189 and 274; 105,190 and 275; 106,191 and 276; 107,192 and 277; 108,193 and 278; 109,194 and 279; 110,195 and 280; 111,196 and 281; 112,197 and 282; 113,198 and 283; 114,199 and 284; 115,200 and 285; 116,201 and 286; 117,202 and 287; 118,203 and 288; 119,204 and 289; 120,205 and 290; 121,206 and 291; 122,207 and 292; 123,208 and 293; 124,209 and 294; 125,210 and 295; 126,211 and 296; 127,212 and 297; 128,213 and 298; 129,214 and 299; 130,215 and 300; 131,216 and 301; 132,217 and 302; 133,218 and 303; 134,219 and 304; 135,220 and 305; 136,221 and 306; 137,222 and 307; 138,223 and 308; 139,224 and 309; 140,225 and 310; 141,226 and 311; 142,227 and 312; 143,228 and 313; 144,229 and 314; 145,220 and 315; 146,231 and 316; And 147,232 and 317.

In other embodiments, antibody or its Fab comprise and are selected from respectively following HCDR3, HCDR2 and HCDR1 aminoacid sequence: SEQ ID NO:1,2 and 3; 1,2 and 34; 1,3 and 35; 1,4 and 35; 1,5 and 36; 1,6 and 36; 1,3 and 36; 1,7 and 36; 1,8 and 36; 1,9 and 36; 1,10 and 38; 1,2 and 38; 1,11 and 39; 1,12 and 40; 1,13 and 41; 1,13 and 42; 1,13 and 33; 1,14 and 43; 1,14 and 44; 1,15 and 45; 1,16 and 45; 1,17 and 46; 1,18 and 47; 1,19 and 47; 1,20 and 48; 1,2 and 49; 1,21 and 50; 1,2 and 51; 1,2 and 52; 1,2 and 53; 1,22 and 54; 1,23 and 55; 1,24 and 56; 1,25 and 46; 1,26 and 57; 1,27 and 58; 1,28 and 59; 1,29 and 60; 1,30 and 61; 1,31 and 62; And 1,32 and 62, and be selected from respectively following LCDR3, LCDR2 and LCDR1 aminoacid sequence: SEQ ID NO:63,148 and 233; 64,149 and 234; 65,150 and 235; 66,151 and 236; 67,152 and 237; 68,153 and 238; 69,154 and 239; 70,155 and 240; 71,156 and 241; 72,157 and 242; 73,158 and 243; 741,159 and 244; 75,160 and 245; 76,161 and 246; 77,162 and 247; 78,163 and 248; 79,164 and 249; 80,165 and 250; 81,166 and 251; 82,167 and 252; 83,168 and 253; 84,169 and 254; 85,170 and 255; 86,171 and 256; 87,172 and 257; 88,173 and 258; 89,174 and 259; 90,175 and 260; 91,176 and 261; 92,177 and 262; 93,178 and 263; 94,179 and 264; 95,180 and 265; 96,181 and 266; 97,182 and 267; 98,183 and 268; 99,184 and 269; 100,185 and 270; 101,186 and 271; 102,187 and 272; 103,188 and 273; 104,189 and 274; 105,190 and 275; 106,191 and 276; 107,192 and 277; 108,193 and 278; 109,194 and 279; 110,195 and 280; 111,196 and 281; 112,197 and 282; 113,198 and 283; 114,199 and 284; 115,200 and 285; 116,201 and 286; 117,202 and 287; 118,203 and 288; 119,204 and 289; 120,205 and 290; 121,206 and 291; 122,207 and 292; 123,208 and 293; 124,209 and 294; 125,210 and 295; 126,211 and 296; 127,212 and 297; 128,213 and 298; 129,214 and 299; 130,215 and 300; 131,216 and 301; 132,217 and 302; 133,218 and 303; 134,219 and 304; 135,220 and 305; 136,221 and 306; 137,222 and 307; 138,223 and 308; 139,224 and 309; 140,225 and 310; 141,226 and 311; 142,227 and 312; 143,228 and 313; 144,229 and 314; 145,220 and 315; 146,231 and 316; And 147,232 and 317.

In other embodiments, antibody or its Fab comprise at least one in the VH aminoacid sequence shown in SEQ ID NO:318-368.

In other embodiments, antibody or its Fab comprise at least one in the VL aminoacid sequence shown in SEQ ID NO:369-453.

In other embodiments, antibody or its Fab comprise the VH region amino acid sequence shown in SEQ ID NO:318,321 or 360, and it is paired with the VL region amino acid sequence that is selected from SEQ ID NO:369-453.

In certain embodiments, antibody or its Fab comprise one or more and are selected from the cdr amino acid sequence of SEQ ID NO:1-317, and wherein said one or more CDR region amino acid sequence comprises at least one or more conserved amino acids replace (for example 1,2,3,4 or 5 conserved amino acid replaces).Conserved amino acid replaces and comprises that class of amino acid is through same class aminoacid replacement, wherein a class is by give a definition: the height in common physical chemistry amino acid side chain character and the being seen homologous protein of occurring in nature replaces frequency, for example, determine by standard Dayhoff frequency switching matrix or BLOSUM matrix.Six large amino acid side chains are sorted out and it comprises: I class (Cys); II class (Ser, Thr, Pro, Ala, Gly); III class (Asn, Asp, Gln, Glu); IV class (His, Arg, Lys); V class (Ile, Leu, Val, Met); And VI class (Phe, Tyr, Trp).For example, replace another III class residue with Asp, replace for conservative such as Asn, Gln or Glu.Therefore, the prediction non-essential amino acid residue in anti-PDGFR β antibody is preferably through of a sort another radical amino acid replacement.Qualification well known in the art is not eliminated the conservative method replacing of amino acid of antigen combination (referring to the people such as such as Brummell, Biochem.32:1180-1187 (1993); The people such as Kobayashi, Protein Eng.12 (10): 879-884 (1999); And the people such as Burks, Proc.Natl.Acad.Sci.USA 94:412-417 (1997)).

In another embodiment, the invention provides anti-PDGFR β antibody or its Fab, its comprise with the VL region amino acid sequence shown in the VH region amino acid sequence shown in SEQ ID NO:318-368 and/or SEQ ID NO:369-453 have respectively approximately 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, VH and/or the VL region amino acid sequence of 98% or 99% identity.

On the other hand, the invention provides the anti-PDGFR β antibody that is incorporated into identical epi-position and/or cross competition with the antibody that comprises the VH domain amino acid sequence shown in SEQ ID NO:318 or its Fab.Such antibody can use conventional competitive binding assay, comprises that for example the competition analysis based on surface plasma body resonant vibration (SPR) is identified.

On the other hand, the invention provides various library in VH territory in pairs, the each member's specific binding of its Chinese library in mankind PDGFR β and wherein diversity in FR1-FR3 district.In a preferred embodiment, the each member in library for example comprises specific binding, in the consistent heavy chain CDR3 of mankind PDGFR β (, the aminoacid sequence shown in SEQ ID NO:1) aminoacid sequence, and wherein diversity in FR1-FR3 district.

On the other hand, the invention provides and stablize various library that VH/VL is right, the each member of its Chinese library is incorporated into mankind PDGFR β.The each member in library preferably comprises VH territory, and described VH territory comprises the CDR3 aminoacid sequence shown in SEQ ID NO:1.Can select to stablize VH/VL couple by any method as known in the art, those shown in the U.S. Provisional Patent Application case 61/453,106 that the mode that described method is including but not limited to quote is in full incorporated herein.

The VH of any type or VL territory expression library all can be used in method of the present invention.Applicable expression library is including but not limited to nucleic acid displaying, phage display and cell surface display library (for example yeast, Mammals and bacterial cell).In a preferred embodiment, library is the nucleic acid display libraries that the method shown in the WO2010/011944 being incorporated herein according to the mode of quoting in full produces.The method of screening expression library well known in the art.Referring to for example Antibody Engineering:Methods and Protocols.Methods in Molecular Biology the 248th volume, (B.K.C.Lo, Ed) Humana Press, 2004 (ISBN:1-58829-092-1), its mode of quoting is in full incorporated herein.

III. modified Binding peptide

In certain embodiments, Binding peptide of the present invention can comprise one or more of modifications.The Binding peptide of the present invention of modified forms can use any technology manufacture as known in the art.

I) reduce immunogenicity

In certain embodiments, use the technology of this area accreditation to modify Binding peptide of the present invention (for example antibody or its Fab) to reduce its immunogenicity.For example, antibody or its fragment can and/or be removed immunization through chimericization, peopleization.

In one embodiment, antibody of the present invention or its Fab can be chimeric.Chimeric antibody is the antibody that the different piece of antibody is derived from different animals species, is derived from the variable region of mouse monoclonal antibody and the antibody of human immunoglobulin constant region such as having.The method of known manufacture chimeric antibody or its fragment in this area.Referring to for example Morrison, Science 229:1202 (1985); The people such as Oi, BioTechniques 4:214 (1986); The people such as Gillies, J.Immunol.Methods 125:191-202 (1989); United States Patent (USP) the 5th, 807, No. 715; The 4th, 816, No. 567; And the 4th, 816, No. 397, its mode of quoting is in full incorporated herein.For manufacturing " chimeric antibody " technology of developing (people such as Morrison, Proc.Natl.Acad.Sci.81:851-855 (1984); The people such as Neuberger, Nature 312:604-608 (1984); The people such as Takeda, Nature 314:452-454 (1985)) can be used for synthetic described molecule.For example, the genetic sequence of the anti-PDGFR β of the mouse antibody molecule of coding binding specificity can merge with the sequence with suitable bioactive human antibody molecule.As used herein, chimeric antibody is the molecule that different piece is derived from different animals species, is derived from the variable region of mouse monoclonal antibody and the molecule of human immunoglobulin constant region, for example humanized antibodies such as having.

In another embodiment, antibody of the present invention or its antigen-binding portion thereof are through peopleization.Humanized antibodies has and comprises from one or more complementary determining region (CDR) of non-human antibody and from the binding specificity of human antibody molecule's framework region.Framework residue in mankind framework region often replaces to change through the corresponding residue of CDR donor antibody, preferably improves antigen combination.Such framework replaces to be identified by method well known in the art, for example, identified for antigen and be combined important framework residue by modeling CDR and the interaction of framework residue, and carry out sequence relatively to identify the uncommon framework residue at specific position.(referring to the people such as such as Queen, United States Patent (USP) the 5th, 585, No. 089; The people such as Riechmann, Nature 332:323 (1988), its mode of quoting is in full incorporated herein.) antibody can use various technology humanization as known in the art, described technology comprises that for example CDR transplants (EP 239,400; PCT publication number WO 91/09967; United States Patent (USP) the 5th, 225, No. 539; The 5th, 530, No. 101; And the 5th, 585, No. 089), facial ornament (veneering) or surface reforming (resurfacing) (EP592,106; EP 519,596; Padlan, Molecular Immunology 28 (4/5): 489-498 (1991); The people such as Studnicka, Protein Engineering 7 (6): 805-814 (1994); Roguska. wait people, PNAS91:969-973 (1994)) and chain reorganization (United States Patent (USP) the 5th, 565, No. 332).

In some embodiments, removing immunization can for example, in order to reduce the immunogenicity of PDGFR β Binding peptide (antibody or its antigen-binding portion thereof).As used herein, term " removal immunization " comprises that change polypeptide (for example antibody or its antigen-binding portion thereof) is to modify t cell epitope (referring to for example WO9852976A1, WO0034317A2).For example, VH and the VL sequence of the initial PDGFR β specific antibody of the present invention or its antigen-binding portion thereof can be analyzed, and the human T cells epi-position " figure " that shows the epi-position position relevant with complementary determining region in sequence (CDR) and other Key residues can be produced from each V district.Analyze the substituting aminoacid replacement with qualification from indivedual t cell epitopes of t cell epitope figure with the final antibody activity of low-risk change.Design a series of substituting VH and VL sequences that comprise aminoacid replacement combination, and subsequently these sequences are incorporated in a series of PDGFR β specific antibodies or its fragment for diagnosis disclosed herein and methods for the treatment of, then test its function.Conventionally produce and the anomaly antibody of test between 12 and 24.Then the complete heavy and light chain gene that comprises modified V and mankind C district is cloned in expression vector and by follow-up plasmid and introduces in clone to manufacture whole antibody (whole antibody).Then in suitable biological chemistry and bioanalysis, compare antibody, and identify best varient.

Ii) effector function and Fc modify

Binding peptide of the present invention can comprise the antibody constant region (for example IgG constant region, for example IgG constant region, for example IgG 1 or IgG4 constant region) that mediates one or more of effector functions.For example, the C1 component of complement be incorporated into antibody constant region can complement activation system.In the conditioning and dissolving of cytopathy substance, complement activation is important.Complement activation also stimulates Inflammatory response and also can relate to autoimmunization allergy.In addition, antibody is incorporated into the acceptor on various cells via Fc district, and wherein the Fc receptor binding site in antibody Fc district is incorporated into the Fc acceptor (FcR) on cell.Exist many inhomogeneity antibody to be had to specific Fc acceptor, comprise IgG (γ acceptor), IgE (epsilon receptor), IgA (α acceptor) and IgM (μ acceptor).The Fc acceptor of antibodies on cell surface causes many important and various biological respinses, comprise by killer cell (being called antibody-dependent cell-mediated cytotoxicity or ADCC) and engulf and destroy the coated particle of antibody, removing immunocomplex, the coated target cell of lytic antibody, discharge inflammatory mediator, placenta transfer and control immunoglobulin (Ig) generation.In preferred embodiments, Binding peptide of the present invention (for example antibody or its Fab) is incorporated into Fc-γ acceptor.In alternate embodiment, Binding peptide of the present invention can comprise and lack one or more of effector functions (for example ADCC activity) and/or can not be in conjunction with the constant region of Fc γ acceptor.

Certain embodiments of the present invention comprise anti-PDGFR β antibody, wherein at least one amino acid in one or more constant region has lacked or has changed to required biochemical characteristics is provided in addition, such as in the time comparing with about identical immunogenic unaltered whole antibody, the effector function that reduces or strengthen, the ability of non-covalent dimerization, the ability raising that is positioned at tumor sites, serum half-life minimizing or serum half-life increase.For example, be territory disappearance antibody for some antibody or its fragment of diagnosis as herein described and methods for the treatment of, it comprises the polypeptide chain that is similar to heavy chain immunoglobulin, but lacks at least a portion in one or more heavy chain territory.For example, in some antibody, a complete territory of the constant region of modified antibody will lack, and all or part of of for example CH2 territory will lack.

In some other embodiment, Binding peptide comprises the constant region (for example, from two or more constant regions in IgG 1, IgG2, IgG3 or IgG4) that is derived from different antibodies homotype.In other embodiments, Binding peptide comprises chimeric hinge (that is hinge of the hinge fraction that comprises the hinge territory (the upper hinge territory of for example IgG4 molecule and IgG1 middle hinge territory) that is derived from different antibodies homotype).In one embodiment, Binding peptide comprises IgG 4 molecule Fc districts or its part, and Ser228Pro in the core hinge area of molecule sudden change (EU numbering).

In certain embodiments, can use technology as known in the art to make the sudden change of Fc part to increase or to reduce effector function.For example, the disappearance in constant region or do not activate (via point mutation or other means) and can reduce the Fc receptors bind of modified antibody of circulation (circulating), increases tumor-localizing thus.In other cases, constant region according to the invention is modified and may be made complement in conjunction with mitigation, therefore reduces serum half-life and non-specific being connected of zygoneure toxin.But other modification of constant region can be in order to modify disulfide linkage or oligosaccharides part, it allows location enhancing because of antigen-specific or flexible increase.Gained physiology overview, biological usability and other biochemical action (such as tumor-localizing, bio distribution and serum half-life) of modifying can be used knows immunological technique measurement and quantitative easily invariably when experiment in the situation that.

In certain embodiments, in antibody of the present invention, Fc territory used is Fc varient.As used herein, term " Fc varient " refers to have with respect to the wild-type Fc territory that produces described Fc territory the Fc territory of at least one aminoacid replacement.For example, wherein Fc territory is derived from IgG 1 antibody, and the Fc varient in described IgG 1Fc territory comprises at least one aminoacid replacement with respect to described Fc territory.

The aminoacid replacement of Fc varient can be positioned at any position, Fc territory (that is the conventional amino acid position of any EU).In one embodiment, Fc varient comprises replacement at the amino acid position that is positioned at hinge territory or its part.In another embodiment, Fc varient comprises replacement at the amino acid position that is positioned at CH2 territory or its part.In another embodiment, Fc varient comprises replacement at the amino acid position that is positioned at CH3 territory or its part.In another embodiment, Fc varient comprises replacement at the amino acid position that is positioned at CH4 territory or its part.

Binding peptide of the present invention can adopt the Fc varient of any this area accreditation, known its effector function and/or FcR in conjunction with aspect give improvement (for example reduce or strengthen).Described Fc varient can comprise for example with lower disclosed arbitrary aminoacid replacement: International PCT is announced WO88/07089A1, WO96/14339A1, WO98/05787A1, WO98/23289A1, WO99/51642A1, WO99/58572A1, WO00/09560A2, WO00/32767A1, WO00/42072A2, WO02/44215A2, WO02/060919A2, WO03/074569A2, WO04/016750A2, WO04/029207A2, WO04/035752A2, WO04/063351A2, WO04/074455A2, WO04/099249A2, WO05/040217A2, WO05/070963A1, WO05/077981A2, WO05/092925A2, WO05/123780A2, WO06/019447A1, WO06/047350A2 and WO06/085967A2 or United States Patent (USP) the 5th, 648, No. 260, the 5th, 739, No. 277, the 5th, 834, No. 250, the 5th, 869, No. 046, the 6th, 096, No. 871, the 6th, 121, No. 022, the 6th, 194, No. 551, the 6th, 242, No. 195, the 6th, 277, No. 375, the 6th, 528, No. 624, the 6th, 538, No. 124, the 6th, 737, No. 056, the 6th, 821, No. 505, the 6th, 998, No. 253, and the 7th, 083, No. 784, it is incorporated herein by reference separately.In an exemplary embodiment, Binding peptide of the present invention can be included in the Fc varient that for example, contains aminoacid replacement on EU position 268 (H268D or H268E).In another exemplary embodiment, Binding peptide of the present invention can for example, for example, contain aminoacid replacement on EU position 239 (S239D or S239E) and/or EU position 332 (I332D or I332Q).

In certain embodiments, Binding peptide of the present invention can comprise Fc varient, and this Fc varient comprises the antigen independence effector function that changes antibody, particularly changes the aminoacid replacement of the circulating half-life of Binding peptide.When compared with lacking the Binding peptide of these replacements, such Binding peptide shows the combination of FcRn increased or reduced, and therefore the transformation period in serum increases respectively or reduces.Expection has and FcRn is improved to the Fc varient of affinity has longer serum half-life, and has applicable application in the mammiferous method for the treatment of of such molecule administration of antibodies has needing long half-lift, for example, with treatment of chronic diseases or illness.On the contrary, the Fc varient that expection FcRn binding affinity reduces has more short-half-life, and such molecule is also applicable to for example to administration, wherein shortening cycling time may be favourable, for example, so that diagnosing image in vivo, or initial antibody has the situation of toxic side effects in the time being present in circulation over a long time.The Fc varient that FcRn binding affinity reduces also unlikely passes placenta, the disease or the illness that are therefore also applicable to treat pregnant woman.In addition, may need other application of the FcRn binding affinity reducing to comprise those application that need brain, kidney and/or liver location.In an exemplary embodiment, of the present invention for example, through changing Binding peptide (antibody or its Fab) demonstration conveying minimizing through glomerular epithelium from vascular system.In another embodiment, of the present invention for example, through changing Binding peptide (antibody or its Fab) demonstration conveying minimizing through blood-brain barrier (BBB) intravasation space from brain.In one embodiment, the antibody that has a FcRn combination of change is included in the Fc territory in " the FcRn coupling collar " in Fc territory with one or more aminoacid replacement.FcRn coupling collar comprises amino-acid residue 280-299 (according to EU numbering).The International PCT being incorporated herein by reference is announced and is openly changed the exemplary aminoacid replacement of FcRn in conjunction with activity for No. WO05/047327.In some exemplary embodiment, Binding peptide of the present invention (for example antibody or its Fab) comprises the Fc territory with one or more following replacement: V284E, H285E, N286D, K290E and S304D (EU numbering).

In other embodiments, there is constant region for the Binding peptide of diagnosis as herein described and methods for the treatment of, for example IgG1 or IgG4 CH, it is through changing to reduce or eliminate glycosylation.For example, Binding peptide of the present invention (for example antibody or its Fab) also can comprise Fc varient, and this Fc varient comprises the glycosylated aminoacid replacement that changes antibody Fc.For example, described Fc varient can have the glycosylation (for example N connects or O connects glycosylation) of minimizing.In exemplary embodiment, Fc varient comprises the glycosylated N of minimizing and connects glycan, and it sees amino acid position 297 (EU numbering) conventionally.In another embodiment, antibody for example, near glycosylation motif (N that contains aminoacid sequence NXT or NXS connects glycosylation motif) or inside there is aminoacid replacement.In one embodiment, antibody is included in amino acid position 228 or 299 (EU numbering) and has the Fc varient of aminoacid replacement.In a more particular embodiment, antibody comprises IgG1 or IgG4 constant region, and this constant region comprises S228P and T299A sudden change (EU numbering).

The International PCT being incorporated herein by reference is announced and is openly given the glycosylated exemplary aminoacid replacement that reduces or change for No. WO05/018572.In preferred embodiments, modify antibody of the present invention or its fragment to eliminate glycosylation.Such antibody or its fragment can be described as " sugar based (agly) " antibody or its fragment (for example " sugar based " antibody).Although not bound by theory, thinks that " sugar based " antibody or its fragment can have improved in vivo security and stability overview.Sugar based (aglycosylated) the Fc district that exemplary sugar based antibody or its fragment comprise IgG4 antibody, it lacks Fc effector function, and the normal vital organ of eliminating thus expressing PDGFR β causes Fc to mediate the possibility of property toxicity.In other embodiments, antibody of the present invention or its fragment comprise the glycan through changing.For example, antibody can have at the Asn297 place in N glycan Shang Fc district the trehalose residue of number of minimizing, that is through without mycose-base (afucosylated).In another embodiment, antibody can have at the Asn297 place in N glycan Shang Fc district the sialic acid residues that changes number.

Iii) covalently bound

Can modify as follows Binding peptide of the present invention: for example make molecule be covalently attached to Binding peptide so that the covalently bound Binding peptide specific binding that can not stop in its homology epi-position.For example; but unrestricted, antibody of the present invention or its fragment can be by glycosylations, acetylize, Pegylation, phosphorylation, amidation, cut, be connected in cell ligand or other oroteins etc. and modify by derivative, the proteolysis of known protection/obstructs base.In numerous chemically modifieds, any all can be undertaken by known technology, and described known technology includes, but is not limited to specificity chemical chop, acetylize, formylation etc.In addition, derivative can contain one or more nonclassical amino acid.

Binding peptide of the present invention (for example antibody or its fragment) can further be held restructuring fusion or engage (comprising covalency and non-covalent joint) with polypeptide or other component chemical at N end or C with heterologous polypeptide.For example, anti-PDGFR β antibody can be suitable for do to detect analyze in the molecule of mark and effector molecule (such as heterologous polypeptide, medicine, radionuclide or toxin) restructuring merge or engage.Announce WO 92/08495 referring to for example PCT; WO 91/14438; WO 89/12624; United States Patent (USP) the 5th, 314, No. 995; And EP 396,387.

Can use method as known in the art to make Binding peptide and heterologous polypeptide merge to increase in vivo the transformation period or for immunoassay.For example, in one embodiment, PEG can engage to increase its transformation period in vivo with Binding peptide of the present invention.Leong, the people such as S.R., Cytokine 16:106 (2001); Adv.in Drug Deliv.Rev.54:531 (2002); Or the people such as Weir, Biochem.Soc.Transactions30:512 (2002).

In addition, Binding peptide of the present invention can merge to contribute to its purifying or detection with flag sequence (such as peptide).In preferred embodiments, marker amino acid sequence is six Histidine peptides, and such as the label for providing in pQE carrier (QIAGEN company, 9259Eton Avenue, Chatsworth, Calif., 91311), wherein many is commercially available.As people such as Gentz, described in Proc.Natl.Acad.Sci.USA86:821-824 (1989), for example, six Histidines are the purifying that fusion rotein facilitates.Other peptide tag that is applicable to purifying includes, but is not limited to " HA " label, and it is corresponding to the epi-position (people such as Wilson, Cell37:767 (1984)) that is derived from influenza hemagglutination fibroin, and " flag " label.

Binding peptide of the present invention can be disengaged form use or can at least one engages with various molecules, for example with the therapeutic property that improves molecule to contribute to target detection, or for patient's imaging or therapy.Binding peptide of the present invention can purifying before or after, in the time carrying out purifying through mark or joint.Particularly, Binding peptide of the present invention can engage with therapeutical agent, prodrug, peptide, protein, enzyme, virus, lipid, biological response modifier, medical agent or PEG.

The Binding peptide of the present invention that is engaged to diagnostic reagent or therapeutical agent is further contained in the present invention.Binding peptide can diagnostic mode for example, in order to for example to monitor the development of immunocyte illness (CLL) or progress (as the part of clinical trial program) for example to determine set effect that treats and/or prevents scheme.Make Binding peptide and detectable substance coupling can contribute to detect.The example of detectable substance comprises various enzymes, prothetic group, fluorescent material, luminescent material, bioluminescent material, radio active material, positron emitting metal (using various positron emission tomographies) and on-radiation paramagnetic metal ion.Referring to for example United States Patent (USP) the 4th, 741, No. 900 about the metal ion that can engage with the antibody that is suitable for do diagnosis thing according to the present invention.The example that is applicable to enzyme comprises horseradish peroxidase, alkaline phosphatase, beta-galactosidase enzymes or acetylcholinesterase; The example that is applicable to prothetic group mixture comprises streptavidin/vitamin H and avidin/biotin; The example that is applicable to fluorescent material comprises umbrella ketone, fluorescein, fluorescein isothiocyanate, rhodamine (rhodamine), dichlorotriazine base amine (dichlorotriazinylamine) fluorescein, dansyl chloride or phycoerythrobilin; The example of luminescent material comprises lumen promise (luminol); The example of bioluminescent material comprises luciferase, fluorescein and aequorin (aequorin); And the example of applicable radio active material comprises 125I, 131I, 111In or 99Tc.

Binding peptide for diagnosis disclosed herein and methods for the treatment of can engage with following: cytotoxin (such as radio isotope, cytotoxicity medicine or toxin) therapeutical agent, cytostatic agent, biotoxin, prodrug, peptide, protein, enzyme, virus, lipid, biological response modifier, medical agent, immunocompetence part (for example lymph medium or other antibody, wherein gained molecule is incorporated into neoplastic cell and effector cell, such as T cell) or PEG.

In another embodiment, for the anti-PDGFR β antibody of diagnosis disclosed herein and methods for the treatment of can with the molecular bond that reduces growth of tumour cell.In other embodiments, disclosed composition can comprise and antibody or its fragment of medicine or prodrug coupling.Other embodiment of the present invention comprises and specific biological body toxin or its cytotoxin fragment (such as ricin (ricin), Bai Shusu (gelonin), Pseudomonas Exotoxin (Pseudomonas exotoxin) or diphtheria toxin) antibody engaging or the purposes of its fragment.Selecting which kind of joint or disengaged antibody to be used will for example, depending on use and patient's symptom of type of cancer and stage, assisting therapy (chemotherapy or external radiation).Should be appreciated that, those skilled in the art can carry out this selection easily in view of enlightenment herein.

Should be appreciated that, in previously research, with isotope-labeled anti-tumour antibody success in order to destroy in animal model and the tumour cell in the mankind in some cases.Exemplary radio isotope comprises: 90Y, 125I, 131I, 123I, 111In, 105Rh, 153Sm, 67Cu, 67Ga, 166Ho, 177Lu, 186Re and 188Re.Radionuclide works by producing ionizing rays, and ionizing rays causes multichain fracture in core DNA, thereby makes necrocytosis.Common high energy α or the beta-particle with short path length of producing of isotropic substance engaging in order to produce therapeutic.Such radionuclide kills its next-door neighbour's cell, the neoplastic cell that for example joiner has connected or entered.It has few impact to delocalization cell or without impact.Radionuclide is non-immunogenic substantially.

IV. the expression of Binding peptide

Operating as mentioned above through separating genetic material to provide after Binding peptide of the present invention, conventionally gene being inserted can be in order to produce in the desired antibody of aequum or the host cell of its fragment to introduce in expression vector.

Term " carrier " or " expression vector " are in this article for the object of specification sheets and claim, and the meaning is the carrier used according to the present invention, and it is as required gene being introduced in cell and at the vehicle of cells.As known in the art, such carrier can be selected from plasmid, phage, virus and retrovirus easily.Generally speaking, the carrier compatible with the present invention is by comprising selective marker, suitably restriction site to be to contribute to cloning required gene and to enter eucaryon or prokaryotic cell prokaryocyte and/or the ability that copies therein.

Numerous expression vector systems can be used for object of the present invention.For example, the carrier utilization of one class is derived from the DNA element of animal virus, and described animal virus is such as being ox papillary tumor virus, polyomavirus, adenovirus, variola virus, baculovirus, retrovirus (RSV, MMTV or MOMLV) or SV40 virus.Other relates to the polycistron system with internal ribosome binding site that uses.In addition, can allow to select to select DNA to be integrated in the cell in its karyomit(e) through the mark of transfection host cell by introducing one or more.Mark can provide auxotrophy host's prototroph, biocide resistance (for example microbiotic) or preventing from heavy metal (such as copper) property.Selectable marker gene can be connected directly to DNA sequence dna to be expressed, or introduces in same cell by cotransformation.Also may need other element to synthesize best mRNA.Such element can comprise signal sequence, splicing signal and transcripting promoter, strengthening and termination signal.In especially preferred embodiment, clone's variable region gene and described above synthetic heavy chain and constant region of light chain gene (being preferably the mankind) are inserted in expression vector.

In other preferred embodiment, Binding peptide of the present invention or its fragment can be expressed with polycistron construct.In such expression system, can make from single polycistron construction body such as the heavy chain of antibody and the target polygene product of light chain.Such system is advantageously used internal ribosome entry site (IRES) so that relatively high-caliber polypeptide of the present invention to be provided in eukaryotic host cell.The United States Patent (USP) being incorporated herein the 6th, open consistency IRES sequence in 193, No. 980.It will be understood by a person skilled in the art that, such expression system can be in order to effectively to make disclosed a whole set of polypeptide in the application's case.

More generally, once prepare the DNA sequence dna of carrier or encoding antibody or its fragment, expression vector can be introduced in suitable host cell.That is, can transformed host cell.Can realize plasmid is introduced in host cell by various technology well known to those skilled in the art.It includes, but is not limited to transfection (comprising electrophoresis and electroporation), protoplast fusion, calcium phosphate Shen Dian, with coating DNA cytogamy, microinjection and infect with intact virus.Referring to Ridgway, A.A.G. " Mammalian Expression Vectors " the 24.2nd chapter, 470-472 page, Vectors, Rodriguez and Denhardt compile, (Butterworths, Boston, Mass.1988).Most preferably, via electroporation, plasmid is introduced in host.Transformant is grown under the condition that is suitable for producing light chain and heavy chain, and it is synthetic to analyze heavy chain and/or light chain protein matter.Exemplary analytical technology comprises enzyme-linked immunosorbent analytical method (ELISA), radioimmunoassay (RIA) or fluorescent activation cell sorter analysis (FACS), immunohistochemistry etc.

As used herein, term " conversion " should use to refer to DNA is introduced to acceptor host cell with broad sense, and it changes genotype, is therefore created in the variation in recipient cell.

According to such same thought, " host cell " refers to the cell that uses the carrier of recombinant DNA technology structure and at least one heterologous gene of encoding to transform.In the description of the method for recombinant host isolated polypeptide, unless clear and definite regulation in addition, term " cell " is used interchangeably to represent antibody sources with " cell culture ".In other words, reclaim polypeptide from " cell " and can mean from of short duration centrifugal full cell, or certainly contain the cell culture recovery of substratum and suspension cell.

In one embodiment, there is Mammals source for the host cell cording of antibody expression; Those skilled in the art can determine concrete host cell system, and it is suitable for required gene product expression most in wherein.Exemplary host cell is to include, but is not limited to DG44 and DUXB11 (Chinese hamster ovary line (Chinese Hamster Ovary line), DHFR-), HELA (human cervical cancer), CVI (monkey-kidney cells system), COS (thering is the CVI derivative of SV40T antigen), R1610 (Chinese hamster fibroblast) BALBC/3T3 (mouse fibroblast), HAK (hamster kidney cell line), SP2/O (mouse myeloma), BFA-1c1BPT (bovine aortic endothelial cells), RAJI (Human Lymphocytes), 293 (mankind's kidneys).In one embodiment, clone provide thus the antibody of expressing through changing glycosylation, for example, for example, without mycose-base (PER.C6.RTM. (Crucell) or (the Potelligent.RTM.Cells) (Biowa of FUT8 gene knockout Chinese hamster ovary celI system, Princeton, N.J.)).In one embodiment, can use NS0 cell.Chinese hamster ovary celI is especially preferred.Host cell system conventionally can be available from commerce services, tissue culture collecting center of the U.S. (American Tissue Culture Collection) or available from open source literature.

External manufacture allows expansion scale to obtain a large amount of required polypeptide.The technology of carrying out mammaliancellculture under conditions of tissue culture is known in this area and comprises homogeneous suspension culture, for example, in airlift reactor or in continuous-stirring reactor, fixing or in enter the cell culture of (entrap), for example in tubular fibre, microcapsule, on Agarose microbead or ceramic cylinder.Necessary and/or while needing, polypeptide solution can pass through usual chromatography method purifying, and described method is for example gel-filtration, ion exchange chromatography, DEAE-Mierocrystalline cellulose chromatography and/or (immunity) affinity chromatography.

The gene of Binding peptide of the present invention or its fragment of encoding also can be the nonmammalian cell of expression, such as bacterium or yeast or vegetable cell.Thus, should be appreciated that various unicellular nonmammalian microorganisms (such as bacterium) also can be converted; That is those can grow or ferment in culture.The bacterium easily transforming comprises Cordycepps (enterobacteriaceae) member in intestines, the bacterial strain such as following: intestinal bacteria (Escherichia coli) or Salmonellas (Salmonella); Bacillaceae (Bacillaceae), such as Bacillus subtilus (Bacillus subtilis); Streptococcus pneumoniae (Pneumococcus); Suis (Streptococcus) and hemophilus influenzae (Haemophilus influenzae).Should be further appreciated that, in the time expressing, polypeptide becomes the part of inclusion body in bacterium.Polypeptide must and then be assembled in functional molecular through separation, purifying.

Except prokaryotic organism, also can use eukaryotic microorganisms.Saccharomyces cerevisiae (Saccharomyces cerevisiae) or the common yeast (baker's yeast) that cures are the most frequently used in eukaryotic microorganisms, but common available many other bacterial strains.For expressing in yeast, conventional plasmid YRp7, for example (people such as Stinchcomb, Nature, 282:39 (1979); The people such as Kingsman, Gene, 7:141 (1979); The people such as Tschemper, Gene, 10:157 (1980)).This plasmid has contained TRP1 gene, and its yeast mutant for shortage energy for growth in tryptophane (ATCC numbering 44076 or PEP4-1) provide selective marker (Jones, Genetics, 85:12 (1977)).The trpl pathology characterizing as yeast host cell genome exists then by providing effective environment not existing to be grown to detect to transform under tryptophane.

V. the pharmaceutical preparation of Binding peptide and application process

On the other hand, the invention provides the pharmaceutical composition that comprises anti-PDGFR β antibody or its fragment.

The method of preparing antibody of the present invention or its fragment and applying it to experimenter is well known to those skilled in the art or is determined easily by those skilled in the art.The route of administration of antibody of the present invention or its fragment can be oral, parenteral, by sucking or local.As used herein, term is non-comprises that through intestines intravenously, intra-arterial, intraperitoneal, intramuscular, subcutaneous, per rectum or transvaginal use.The non-intravenously of using through intestines, intra-arterial, subcutaneous and intramuscular form are generally preferably.Although all these administration forms are all clearly covered by category of the present invention, administration form will be injection solution, be used in particular for intravenously or intra-arterial injection or drop.The pharmaceutical composition that is applicable to injection can comprise buffer reagent (such as acetate, phosphoric acid salt or citrate buffer agent), interfacial agent (such as polysorbate), stablizer (such as people's albuminoid) etc. optionally conventionally.But, with enlighten herein in other compatible method, polypeptide can directly be passed to the site of unfavorable cell colony, increases thus the exposure of diseased tissue to therapeutical agent.

Comprise sterile aqueous or non-aqueous solution, suspension and emulsion for the non-preparation of using through intestines.The example of non-aqueous solvent is propylene glycol, polyoxyethylene glycol, vegetables oil (such as sweet oil) and injectable organic ester (such as ethyl oleate).Aqueous carrier comprises water, alcohol/aqueous solution, emulsion or suspension, comprises salt solution and buffer medium.In theme invention, pharmaceutically acceptable carrier includes, but is not limited to 0.01-0.1M and preferred 0.05M phosphate buffered saline buffer or 0.8% salt solution.Other is common non-ly comprises sodium radio-phosphate,P-32 solution, woods Ge Shi dextrose (Ringer's dextrose), dextrose and sodium-chlor, lactated Ringer solution through intestines mediator, or fixed oil.Intravenously mediator comprises fluid and nutrient prime replenisher, electrolytic solution supplement, such as the thing based on woods Ge Shi dextrose etc.Also can there is sanitas and other additive, such as biocide, antioxidant, sequestrant and rare gas element etc.More specifically, the pharmaceutical composition that is applicable to injectable purposes comprises that aseptic aqueous solution (wherein water-soluble) or dispersion liquid and aseptic powder are to prepare sterile injectable solution or dispersion liquid temporarily.Under these circumstances, composition must be aseptic and be should be fluid (to the degree that has easy syringeability).Its should manufacture and condition of storage under stable and preferably will avoid such as the microbiological contamination effect of bacterium and fungi and preservation.Carrier can be and contains such as solvent or the dispersion medium of water, ethanol, polyvalent alcohol (such as glycerine, propylene glycol and liquid macrogol etc.) and applicable mixture thereof.Adequate liquidity can be for example by using dressing (such as Yelkin TTS), in dispersion liquid situation by maintaining desired particle size and by maintaining with interfacial agent.Prevent that microbial process from can such as, reach by various antibacterial agents and anti-mycotic agent (p-Hydroxybenzoate, butylene-chlorohydrin, phenol, xitix, second mercury Thiosalicylic acid,sodiumsalt etc.).In many cases, in composition, preferably include isotonic agent, for example sugar, polyvalent alcohol (such as mannitol, Sorbitol Powder) or sodium-chlor.The permanent absorption of Injectable composition can postpone by comprising at composition the reagent (for example aluminum monostearate and gelatin) of absorption and reach.

Under any circumstance, sterile injectable solution all can be prepared as follows: active compound (for example independent antibody or antibody and the combination of other promoting agent) is incorporated in the appropriate solvent with a kind of composition cited herein or its combination with aequum as required, then carries out filtration sterilization.Generally speaking, dispersion liquid is by active compound is incorporated in aseptic mediator and is prepared, and this aseptic mediator contains alkaline dispersion medium and from enumerating those required other composition above.In the case of the aseptic powder for the preparation of sterile injectable solution, preferably preparation method is vacuum-drying and lyophilize, and it obtains activeconstituents and adds the powder from any other required composition of the solution of its previous sterile filtration.Process according to procedures known in the art injection preparation, be filled in the container such as ampoule, sack, bottle, syringe or bottle, and in sealed under aseptic conditions.In addition, preparation can be such as the United States Patent (USP) sequence number the 09/259th coexisting in application, No. 337 and United States Patent (USP) sequence number the 09/259th, kit form packaging and the sale described in No. 338 (it is incorporated herein by reference separately).Such goods are indicated the compositions related experimenter that autoimmune disorder or superfluous natural disposition illness were suffered from or easily suffered from treatment that is applicable to by preferably having mark or package insert.

Treat the stabilization antibody of the present invention of above-mentioned symptom or the effective dose of its fragment and change depending on many different factors, described factor comprises that the means of using, target site, patient physiological state, patient are for the mankind or animal, other medicine of using and treatment are for preventative or curative.Conventionally, patient is the mankind, but also can treat the non-human mammal that comprises transgene mammal.Therapeutic dose can use ordinary method titration well known by persons skilled in the art so that security and effect optimizing.

For carrying out passive immunization with antibody of the present invention, dosage can such as, between such as approximately 0.0001 to 100mg/kg and more generally in the scope of 0.01 to 5mg/kg (0.02mg/kg, 0.25mg/kg, 0.5mg/kg, 0.75mg/kg, 1mg/kg, 2mg/kg etc.) host's body weight.For example, dosage can be 1mg/kg body weight or 10mg/kg body weight or in the scope of 1-10mg/kg, is preferably at least 1mg/kg.Dosage in the middle of above-mentioned scope is also intended in category of the present invention.

Can every day, the next day, weekly or rule of thumb any other time-histories of Analysis deterrmination is used such dosage to experimenter.Exemplary treatment need to be gone through over a long time (for example at least six months) and use multidose.Other exemplary treatment plan needs to use once or use January once or use once for every 3 to 6 months for every two weeks.Exemplary dosage time-histories be included in running days 1-10mg/kg or 15mg/kg, the next day 30mg/kg or 60mg/kg weekly.In certain methods, use the two or more monoclonal antibodies with different binding specificities simultaneously, in this case, the application dosage of each antibody can belong in stated limit.

Antibody of the present invention or its fragment can be used under multiple occasions.Interval between single dose for example can be every day, weekly, monthly or every year.Interval also can be irregular, as shown in the blood level by polypeptide or target molecule in measurement patient body.In certain methods, adjust dosage to reach a certain plasma antibody or toxin concentration, for example 1-1000 μ g/ml or 25-300 μ g/ml.Or antibody or its fragment can be used as extended release preparation and use, need to use with small frequency more in this case.Dosage and frequency changed depending on antibody half life in patient body.Generally speaking, humanized antibodies shows long half-lift, is then chimeric antibody and non-human antibody.In one embodiment, antibody of the present invention or its fragment can be used with disengaged form.In another embodiment, antibody of the present invention can repeatedly be used with joint form.In yet another embodiment, antibody of the present invention or its fragment can be used with disengaged form, then use with joint form, or vice versa.

The visual treatment of application dosage and frequency is preventative or curative variation.In prophylactic application, use the composition that contains antibody of the present invention or its mixture to the patient in morbid state not yet to strengthen patient's resistibility.This amount is defined as " preventative effective dose ".In this purposes, accurately amount is again depending on patient health situation and general immunity, but generally between every dose 0.1 to 25mg, especially in the scope of every dose 0.5 to 2.5mg.With relatively not frequently interval use relatively low dosage and go through distance.Some patients continued to receive treatment in its remaining years.

In therapeutic application, sometimes (for example every dose approximately 1 to 400mg/kg antibody to need relatively high dosage under relatively short interval, wherein 5 to 25mg dosage be more usually used in radiation immunity joiner and more high dosage for cytotoxin-medicine joint molecule) until progression of disease reduce or stop, and preferably until patient shows disease symptoms partially or completely improves.After this, can use Prevention scheme to patient.

In one embodiment, the nucleic acid molecule of available code polypeptide of the present invention (for example, in carrier) treatment experimenter.The dosage of the nucleic acid of coded polypeptide is in the scope of the about 10ng to 1g of each patient, 100ng to 100mg, 1 μ g to 10mg or 30-300 μ g DNA.The dosage of venereal infection poisonous carrier is from every dose of 10-100 or the variation of more virus particle.

Therapeutical agent can be by non-in intestines, surface, intravenously, per os, subcutaneous, intra-arterial, encephalic, intraperitoneal, nose or intramuscular means use so that preventative and/or therapeutic treatment.For using antibody of the present invention, intramuscular injection or intravenous infusion are preferred.In certain methods, by therapeutic antibodies or its fragment direct injection to cranial cavity.In certain methods, with sustained-release composition or device (such as Medipad ^tMdevice) form administration of antibodies or its fragment.

Reagent of the present invention optionally needs the illness for the treatment of (for example preventative or therapeutic) or other agent combination of symptom to use with effective treatment.Preferred other reagent is that this area is approved and standard type is used the reagent for particular condition.

Effective single therapy dosage of the antibody of the present invention of 90Y mark (that is treatment significant quantity) between approximately 5 and about 75mCi between, more preferably between approximately 10 and about 40mCi between scope in.The non-marrow of effective single therapy of the antibody of 131I mark remove property dosage between approximately 5 and about 70mCi between, more preferably in the scope between about 5mCi and about 40mCi.The effective single therapy of the antibody of 131I mark remove property dosage (that is may need autologous bone marrow transplantation) between approximately 30 and about 600mCi between, more preferably between approximately 50 and be less than in the scope between about 500mCi.In conjunction with chimeric modified antibodies, due to the longer circulating half-life with respect to rodent antibody, therefore the non-marrow of effective single therapy of the chimeric antibody of iodine-131 mark remove property dosage between approximately 5 and about 40mCi between, be more preferably less than in the scope of about 30mCi.The imaging criterion of for example 111In mark is less than about 5mCi conventionally.

Although obtained many clinical experiences with 131I and 90Y, other radio-labeling is known and for similar object in this area.Other radio isotope is used for to imaging.For example, other radio isotope compatible with category of the present invention includes, but is not limited to 123I, 125I, 32P, 57Co, 64Cu, 67Cu, 77Br, 81Rb, 81Kr, 87Sr, 113In, 127Cs, 129Cs, 132I, 197Hg, 203Pb, 206Bi, 177Lu, 186Re, 212Pb, 212Bi, 47Sc, 105Rh, 109Pd, 153Sm, 188Re, 199Au, 225Ac, 211A, 213Bi.Thus, α, γ and beta emitter are all compatible with the present invention.In addition,, in view of the disclosure, those skilled in the art think and can invariably when experiment in the situation that, determine that easily which kind of radionuclide is compatible with the selected course for the treatment of.For this purpose, comprise 125I, 123I, 99Tc, 43K, 52Fe, 67Ga, 68Ga for other radionuclide of clinical diagnosis, and 111In.Also with multiple radioisotope labeling antibody to may be used for target immunotherapy (people such as Peirersz, Immunol.Cell Biol.65:111-125 (1987)).Such radionuclide comprises 188Re and 186Re and 199Au and 67Cu with less degree.United States Patent (USP) the 5th, provides for 460, No. 785 about so radioisotopic other data and is incorporated herein by reference.

As previously discussed, antibody of the present invention or its fragment pharmaceutically significant quantity use in vivo treat Mammals illness.Thus, should be appreciated that, disclosed antibody or its fragment will be through allotment to contribute to administering active agents and promote promoting agent stability.Preferably comprise pharmaceutically acceptable nontoxicity sterile carrier according to pharmaceutical composition of the present invention, such as physiological saline, nontoxic buffer reagent, sanitas etc.For purposes of this application, engage with therapeutical agent or the pharmaceutically significant quantity of unassembled antibody of the present invention should to be considered to the meaning be to be enough to reach the amount that is effectively incorporated into target and reaches benefit (for example improving symptom or detection material or the cell of disease or illness).The in the situation that of tumour cell, polypeptide preferably can with neoplastic cell or immunologically competent cell on selected immuno-activated-antigen interact and the increase of those necrocytosiss be provided.Certainly, pharmaceutical composition of the present invention can use to provide the pharmaceutically polypeptide of significant quantity by single or multiple dosage.

Consistent with category of the present disclosure, can use antibody of the present invention with the amount that is enough to produce therapeutic or prophylactic action to the mankind or other animal according to above-mentioned methods for the treatment of.Polypeptide of the present invention can be used to these mankind or other animal by combine regular dosage form prepared by antibody of the present invention and pharmaceutically acceptable conventional carrier or thinner according to known technology.Those skilled in the art will appreciate that the form of pharmaceutically acceptable carrier or thinner and feature specify by knowing parameter with the amount of the activeconstituents of its combination, route of administration and other.Those skilled in the art should be further appreciated that the provable mixture that comprises one or more of peptide materials of the present invention is especially effective.

VI. treat the method for PDGFR ss related diseases or illness

Binding peptide of the present invention or its fragment are applicable to antagonism PDGFR 'beta ' activity.Therefore, on the other hand, the invention provides the method for the treatment of PDGFR ss related diseases or illness by using to the experimenter who has needs the pharmaceutical composition that comprises one or more of anti-PDGFR β antibody of the present invention or its Fab.

PDGFR ss related diseases or the illness of receiving treatment are including but not limited to: the macular degeneration (AMD) that the age is relevant; Restenosis, comprises that arteries operation, congee sample spot surgical blanking (atherectomy) or other patch remove coronary restenosis after intrusion method, and same program metanephros or Peripheral arteries restenosis; Blood vessel hyperplasia phenomenon and the fibrosis relevant to other acute injury form, such as: wait the pulmonary fibrosis of faciation pass, the renal fibrosis relevant with ephritis, the coronary stenosis relevant with mucocutaneous lymphnode syndrome (Kawasake's disease) to adult respiratory distress, and the blood vessel relevant to other arteritis (such as aortic arch syndrom (Takayasha's disease)) narrows; Fibrosis process, such as scleroderma, myofibrosis; And cancer (for example tumor cell proliferation and neovascularity generate)

Those skilled in the art should be able to be identified for by normal experiment effective, the nontoxic amount of the antibody (or other therapeutical agent) of the object for the treatment of PDGFR ss related diseases or illness.For example, the therapeutic activity amount of polypeptide can be according to changing such as following factor: experimenter's disease stage (for example the I stage is with respect to the IV stage), age, sex, medical science complication (for example immunosuppressant symptom or disease) and body weight, and antibody causes the ability of required reaction in subject.Capable of regulating dosage regimen is to provide optimum therapeutic response.For example, can use some fractionated doses every day, or dosage can as specifiedly in the urgency by treatment situation reduce in proportion.But generally speaking, expection effective dose is approximately 0.05 to 100 milligrams of per kilogram of body weight every days and more preferably in the per kilogram of body weight scope of approximately 0.5 to 10 milligrams every day.

Embodiment

The present invention is further illustrated by following instance, described following instance should be considered as being further limited.The content of sequence table, accompanying drawing and all reference, patent and the publication application of quoting in whole the application's case is all clearly incorporated herein by reference.

Embodiment 1: separate specific binding in the VH territory of mankind PDGFR β

Use the DNA as shown in WO2010/011944 (mode of quoting is in full incorporated herein) to show to select specific binding in the VH territory of mankind PDGFR β.Especially, for mankind PDGFR β, carry out six and take turns selection being derived from the primary mankind VH domain dna display libraries of ten bone marrow donors.Clone selected binding substances and to its order-checking.VH territory clone A4, B4 and G2 are selected in screening since then, and its aminoacid sequence is set forth in table 3.

Embodiment 2:HCDR3 reorganization

A.VH library construction

For screening has the territory in conjunction with the improved VH of feature, the HCDR3 sequence reorganization that will clone A4 (being appointed as XB1511) is extremely in primary mankind VH library, for being incorporated into the mankind and mouse PDGFR β further selects it.Especially, the DNA sequence dna of the HCDR3 (SEQ ID NO:1) of composite coding clone A4 and use framework specific oligonucleotide to be assembled to comprise in the library of the framework region 1-3 in the primary mankind VH territory of marrow B cell and PBMC amplification.Carry out amplifying human VH framework region 1-3 to produce indivedual libraries of VH family framework region with 5'VH family specificity and the general FR3 reverse primer of 3'.Reorganize VH family framework library and XB1511HCDR3 by carrying out further pcr amplification with 5'T7TMV and 3'XB1511FR3CDR3FR4 oligonucleotide.This also adds T7TMV promoter sequence so that in-vitro transcription/translation at 5' end.Also by respectively with FR4Cu3 oppositely and Y109 primer and 5'T7TMV primer carry out PCR and add C end C μ 3 sequences and FLAG label (for translating rear purifying).In table 5, set forth the nucleotide sequence for the preparation of the oligonucleotide in HCDR3 reorganization VH library.In Fig. 1, set forth the schematic diagram of VH library construction.

Table 5: for building the oligonucleotide in HCDR3 reorganization VH library

B. library screening

Then HCDR3 reorganized to library, VH territory is transcribed in mRNA library and select with the dsDNA display technique described in WO2010/011944.Select 4 to take turns with the wheel mankind and the mouse PDGFR β that replace.Absorption and dissociation rate in wheel applied dynamics control continuously screen to improve selection preciseness, therefore select PDGFR β to have the VH territory of high-affinity.Particularly, select as follows: the 1st takes turns (R1) with the fixing mankind PDGFR β of 10nM; Fixing 100nM mouse PDGFR β for R2; R3 with 10nM solvable mankind PDGFR β and with the fixing mankind PDGFR β of 200nM competition 24 hours and 120 hours; And 10nM mouse PDGFR β for R4.Subclone R4 in conjunction with pond so that DNA sequencing.R4 shows that in conjunction with the sequential analysis in pond the HCDR3 of XB1511 is present in multiple different framework content.Do not obtain wild-type parent sequence from analyzed sequence set.In table 3, set forth the aminoacid sequence in selected VH territory herein.

C. the binding specificity in selected HCDR3 reorganization VH territory

Use ³⁵in Vitro Translation library assessment selected R4 combination to the mankind and mouse PDGFR β in conjunction with pond above of S Met mark.Particularly, assess the combination of described pond to epoxy resin bead, 100nM IgG, mankind PDGFR β and mouse PDGFR β.As shown in Figure 2, parent XB1511VH territory shows that specific binding is in mankind PDGFR β, and can not detect and be incorporated into mouse PDGFR β.Framework reorganization preliminary election library shows the weak binding to mankind PDGFR β.But on the contrary, R4 framework reorganization library demonstration is significantly incorporated into the mankind and mouse PDGFR β.

Embodiment 3: VL/VH couple is stablized in qualification

A. build VL DNA library

By RT-PCR from the B cell construction mankind VL library of young healthy donor (Allcells) (V κ and V λ).For guaranteeing library diversity, obtain 300,000,000 myelomonocytes and 100,000,000 periphery blood mononuclear cells and for primary VH and VL library construction from ten donors.In Fig. 3, set forth the schematic diagram of library production method.

Described in table 4, be designed for the Oligonucleolide primers of the synthetic and follow-up pcr amplification of the cDNA of V κ and V λ sequence.Particularly, the V κ and the V λFR1 district that certainly have each family of upstream UTR sequence design multiple sense primer.Be designed for the antisense primer of κ and λ gene amplification from the constant region that is nested in C κ 1 (C κ 2) or there is the J λ (J λ C κ 2) in identical C κ 2 downstreams.V κ carries identical C terminal sequence to carry out pcr amplification during selection cycle with V λ library.

The scheme that using FastTrack mRNA to prepare test kit (Invitrogen) provides according to test kit is prepared mRNA from indivedual donors.Use light chain κ and λ constant region (C κ 1 and C λ 1) are had to specific primer synthetic the first chain cDNA of separating mRNA that hangs oneself.

Use cDNA to carry out the pcr amplification of V κ and V λ sequence as template with C κ 2 and V κ family specificity or J λ C κ 2 mixtures and V λ family specificity primer.Indivedual V κ and V λ family and indivedual donor are carried out to PCR and continue 18-20 cycle.After gel-purified, collect from the V κ in variant source and V λ library to produce final V κ and V λ library.

Table 6: for building the oligonucleotide of mankind V λ and V κ DNA display libraries

B. show that by dsDNA producing VL merges library

Use T7Megascript test kit (Invitrogen, catalog number (Cat.No.) AM1334) that the V κ and the V λ DNA library that use the method described in the present embodiment to produce are transcribed in mRNA library.Carry out purified mRNA by the scheme that RNeasy MinElute Cleanup test kit (Qiagen, catalog number (Cat.No.) 74204) provides according to test kit.Described in WO2010/011944,600pmol RNA (300pmol V κ and V λ library) shows that with dsDNA connexon and component engage and assemble altogether.In Vitro Translation is carried out in the VL library of assembling and merge library to produce, wherein each VL territory (phenotype) merges with its encoding sequence (genotype) is stable.Will ³⁵s Met is incorporated in translation process so that fusions is carried out to radio-labeling.This library then by oligomerization dT cellulose purification, use the standard molecular biological technique of reverse transcription to be converted into that dsDNA display libraries, RNaseH decompose, the 2nd chain DNA synthesizes, then carry out flag label purifying.

C. identify the VL couple in XB1511 and XB2202VH territory

XB1511VH territory is translated as to free protein (will ³⁵s Met is incorporated in translation reaction) and carry out affinity purifying via c end flag label.Then mix the fusions library, VL territory (as above preparation) of XB1511VH territory and purifying with equimolar ratio, and at 25 DEG C overnight incubation so that VH and VL fusion area via its hydrophobic flakes external being connected.Then make mixture contact with the PDGFR β target pre-fixing on Epoxy450 bead or in solution, and caught by a-protein bead, washing is incorporated into the mixture of fixing PDGFR β target and with 0.1N KOH wash-out.Carry out PCR by VL Auele Specific Primer group and be incorporated into the VL of PDGFR β target to reclaim, both be all VH-VL to and be VL territory in pairs.Carrying out 3, to take turns VL paired, and front 2 take turns and have low preciseness (100nM PDGFR β) and third round have high preciseness (10nM PDGFR β).Similarly, Yi YuVL library, XB2202VH territory is paired for two-wheeled.Take turns XB2202/VL in pairs and select for each, improving as follows preciseness: by absorption and the dissociation rate strategy of dynamic (dynamical) control, being combined to identify to be stable into right VL territory and to strengthen VH with XB2202VH territory.

Then the pond, VL territory of qualification is above cloned in Blunt Zero TOPO carrier (Invitrogen), and by using M13 forward and reverse primer to carry out PCR from gained bacterium colony amplification coding VL DNA sequence dna.Then individual other checked order through the coding VL DNA sequence dna increasing.The sequence data obtaining from VL pond shows that various spectrum of VL obtains enrichment via the method.Multiple families and framework are present in pond.Some VL exist with duplicate or family's form.Can identify that unique VL family and some VL exist more than once.In table 4, set forth herein use the inventive method qualification be combined the paired exemplary VL sequence of VH territory XB1511 and XB2202 with PDGFR β.

D. assess VH and VL couple through qualification

For assessment is through the right feature of qualification VH-VL, express and build and produce 10-12 the scFV from each pond by In Vitro Translation or by intestinal bacteria (E.coli), then carry out affinity purifying.

Carry out PDGFR β in conjunction with elisa assay with assessment scFv combination and the definite EC50 to fixing PDGFR β.Particularly, at 4 DEG C, 2 μ g/mL mankind PDGFR β in PBS and human Fc or IgG are fixed on Maxisorp plate and are spent the night.Then wash this plate and seal with superblock.In vitro translated thick scFv lysate 1:3 is diluted in 1 × PBST.By 100 μ l dilution scFv lysate loadings to each hole of Maxisorp plate and at room temperature hatch 1 hour.Detect the scFv that is incorporated into fixing PDGFR β by anti-flag antibody-HRP (under 1:5000 extent of dilution) and tmb substrate.Under OD 450nm, carry out reading this plate on the Molecular Device plate reader of end point analysis.As shown in the 4th, 5 and 6 figure, in ELISA binding analysis, in the scFv for XB1511 and XB2202 generation, be greater than 50% demonstration specific binding in PDGFR β.On the contrary, independent in pairs VL do not show and be incorporated into PDGFR β (referring to Fig. 7).

Affinity by the balance based on solution in conjunction with the some scFv of Analysis deterrmination.Particularly the scFv RNA of 120pmol is translated to and had ³⁵in the free protein of S Met.Will be through translation reaction mixture diluted to 3 times in the binding buffer liquid that contains 1 × PBS and 0.025% triton (triton), 1mg/mL BSA and 0.1mg/mL sssDNA.Mankind PDGFR β is diluted in same binding buffer liquid to the ultimate density of 100nM to 0nM.ScFv mixture final volume with 100 μ l together with hPDGFR β of dilution is hatched on Kingfisher plate (Thermofisher Scientific, 97002084).After hatching, use 25 μ l a-protein magnetic beads (Invitrogen) to catch PDGFR β in solution.The PDGFR β that catches of washing and in kingfisher Reader (Thermofisher Scientific) wash-out.Use scintillometer to be incorporated into magnetic beads-fixing hPDGFR β scFv (with ³⁵s Met mark) amount count, and with Graph Pad Prism 5 calculating K d.For tested XB1511 source property scFv, when compared with independent XB1511VH, 2 scFv show high 8-10 Kd doubly, and 1 shows the Kd of high 2.5 times, and 4 show similar Kd (Fig. 8).Only 1 scFv shows the K lower than independent XB1511VH _d.As shown in Figure 9, when compared with independent XB2202VH, the XB2202 source property scFv testing all shows doubly better Kd of about 8-10.

Embodiment 4: the binding affinity of anti-PDGFR β VH territory to the mankind and mouse PDGFR β

By the mankind of selected R4 framework reorganization in embodiment 2 and the pond, VH territory of mouse PDGFR β enrichment is cloned in coli expression carrier, generation and purifying.On Biacore T100, use surface plasma body resonant vibration to determine the binding kinetics of VH territory to the mankind and mouse PDFGR.In brief, use with the Series CM5 sensing wafer (CM5) of anti-hIgG1Fc monoclonal antibody coupling and fix respectively the mankind and mouse PDGFR-hIgG1-Fc chimeric fusion protein.For each cycle, first catch PDGFR fusion rotein, then inject VH115 second (being connected) with the flow rate of 100 μ L/min.After the stage of being connected, be 600 seconds dissociate the stage immediately.Make surface regeneration at each cycle single injection 3M MgCl2 (10 μ L/min, 60 seconds).Inject the VH territory (0.55nM-40nM) of multiple concentration and analyze gained sensing figure with T100Evaluation software.Determine binding kinetics with the matching of 1:1 binding curve.10th, in 11 and 12 figure, show respectively VH territory clone XB2202 and the binding kinetics of XB2708 to the mankind and mouse PDGFR β.Such result demonstration XB2202 and XB2708 affinity compared with parent XB1511 are improved 50-150 doubly.Particularly, the Kd of XB2202 and XB2708 is respectively 249pM and 93pM, and dissociation rate (Koff) is respectively 1.86 × 10 ^-3and 9.267 × 10 ^-4.XB2202 and XB2708 are all incorporated into the mankind and mouse PDGFR β.Although especially note that it shares identical HCDR3, XB2202 is derived from VH1 family reproductive tract sequence and XB2708 is derived from VH3 family reproductive tract sequence.

Embodiment 5: suppress PDGFBB and be bonded to PDGFR β

Use surface plasma body resonant vibration on Biacore T100, to assess XB2202VH disclosed herein territory antagonism PDGFBB ligand binding in the ability of mankind PDFGRb.In brief, use with the Series CM5 sensing wafer of anti-hIgG1Fc monoclonal antibody coupling and fix mankind PDGFR-hIgG1-Fc chimeric fusion protein.10nM mankind PDGFBB is injected in to the mankind PDGFR β catching in advance, obtains and do not have the 100% association reaction unit to PDGFR β under VH.For each consecutive periods, first catch PDGFR fusion rotein, then inject VH territory 120 seconds.Washing out behind unconjugated VH territory, the PDGFBB that then injects 10nM continues 120 seconds.Make surface regeneration at each cycle single injection 3M MgCl2 (10 μ L/min, 60 seconds).Inject the VH (0.46nM-60nM) of multiple concentration, analyze gained sensing figure with T100Evaluation software, and calculate PDGFBB in conjunction with inhibition.As shown in Figure 13, XB2202 is incorporated into mankind PDFGRb with the IC50 inhibition PDGFBB that is less than 5nM.

Embodiment 6: suppress pericyte migration

Determine the ability of the external pericyte migration of XB2708VH disclosed herein territory antagonism PDGF-BB inducibility.Obtain elementary human retina pericyte and cultivate according to manufacturers's suggestion complete growth medium of CSC from Cell Systems Corporation (Kirkland, WA).Approximately 125 cells (2-5 subculture) are inoculated in to 384 holes that scribble human plasma fibronectin (5 μ g/ml are in PBS) in each hole of biological sensing plate, and seal with containing BSA (1% in PBS) in the serum-free medium that contains 0.1%BSA.Allow cell adhesion, then serum is suffered from hunger and is spent the night.After serum is suffered from hunger, together with the VH for mankind PDGFR beta receptor of cell and various concentration is in tissue culture thermostat container, hatch 1 hour.In the time that finishing, antibody preincubate stimulates migration in PDGF-BB is added into serum free medium with the ultimate density of 5ng/ml.In tissue culture thermostat container at 37 DEG C and 5%CO ₂and use under >75% humidity scanner obtains hole image for every 18 minutes and continues 20 hours.Use the barycenter qualification based on Matlab and follow the trail of algorithm and analyze collected data with the cell speed of calculating between 10 hours and 16 hours.Result described in Figure 14 shows the IC50 antagonism PDGF-BB inducibility pericyte migration that XB2708 can 0.54nM.

Embodiment 7:VH-VL is to being converted into allos four poly-IgG and bioactive proofs

XB1511VH expresses together with in the poly-IgG of allos in 293T cell four with D8VL.Collecting cell culture supernatants after 48 hours and 96 hours, and with a-protein agarose beads purify express IgG.IgG with 8mg/L manufacture and without any optimizing.For the biological activity of assessment XB1511/D8IgG, HFF-1 human foreskin fiber parent cell is inoculated in 384 hole BIND biosensors and that it is adhered in serum free medium is overnight.Follow with 5ng/mL or 10ng/mL PDGFBB ligand stimulation inoblast, and it is moved 18 hours in 100nM XB1511/D8IgG existence or not.Within every 15 minutes, catch BIND scanner image and use software analysis tool to measure the course length of indivedual cell migration reactions.Course length is by representing from blue (non-migratory) to red (maximum migration) " thermal map (heat map) ".As shown in Figure 15, XB1511/D8IgG can block the PDGFBB inducibility migration of mankind's fibroblast completely.

Embodiment 8:scFv thermostability

Determine the thermostability of XB2202VH and XB2202/A4scFv.Particularly, at 4 DEG C, 37 DEG C, 60 DEG C and 70 DEG C, hatch 1mg/mL XB2202 and XB2202-A4 continue 12 hours, and carry out PDGFR β in conjunction with ELISA with test protein the combination activity after hatching.As shown in Figure 16, XB2202VH territory loses remarkable PDGFR β in conjunction with activity after hatching at 60 DEG C, and after hatching at 70 DEG C, loses completely in conjunction with active.The Tm of XB2202 measures as approximately 62 DEG C.On the contrary, XB2202/A4scFv was hatched after 12 hours at 70 DEG C has activity completely, shows that the Tm of XB2202scFv is greater than 70 DEG C.

The expression of embodiment 9:IgG1 antibody, purifying and concentrated

XB1511/D8 and XB2202/A4VH/VL to respectively with the poly-IgG1 antibody formation of total length allos four at 293T cells, and purifying in addition.In table 7, set forth XB1511/D8 and the heavy chain of XB2202/A4IgG1 antibody and the aminoacid sequence of light chain herein.

Cell culture supernatant obtains and uses the expressed antibody of two-step purifying flow process purifying by filtration.Particularly, carry out a-protein affinity purifying, binding antibody is at 3.5 times wash-outs of pH.Use 1M Tris that the pH value of a-protein elutant is adjusted to pH 7, and be further purified by the ion exchange chromatography that uses HiTrap Q XL tubing string (GE Healthcare).The antibody of purifying is stored in pH 7PBS.

Table 7: form is XB1511/D8 and the right aminoacid sequence of XB2202/A4VH/VL of total length allos four poly-IgG1 antibody formations.

By measuring the A of antibody-solutions ₂₈₀determine antibody expression level and antibody concentration after each purification step.The purity of the antibody of purifying and quality are determined by size exclusion high performance liquid chromatography (SEC-HPLC).In table 8, set forth the result of such experiment herein.Such data presentation is in the time that XB1511/D8 and XB2202/A4VH/VL are total length allos four poly-IgG1 antibody formation to form, and gained antibody is that height can be manufactured, because its expression level is high, be easily purified to high purity and show few gathering.

The analysis of the Expression and purification of table 8:XB1511/D8 and XB2202/A4IgG1 antibody.

Further analyze the XB1511/D8 of purifying and the concentrating capacity of XB2202/A4IgG1 antibody.Particularly, use and there is the centricon ultrafiltration rotation tubing string that 10kDa and 30kDa block restriction each antibody-solutions is concentrated into 50mg/ml.Analyze the integrity of concentrated solution by SEC-HPLC.The purity of Analysis deterrmination 50mg/ml XB1511/D8IgG1 solution is approximately 96% and containing the 2.4% antibody aggregation body of having an appointment since then, and the purity of 50mg/ml XB2202/A4IgG1 solution is approximately 97.8% and containing having an appointment 2.2% antibody aggregation body.Such digital proof XB1511/D8 and XB2202/A4IgG1 antibody are stable at concentrated solution camber.

The thermostability of embodiment 10:XB1511/D8 and XB2202/A4IgG1 antibody

Use Analysis deterrmination XB1511/D8 based on fluorescence and the thermostability of XB2202/A4IgG1 antibody.Particularly, XB1511/D8IgG1, the XB2202/A4IgG1 of 5mg/ml purifying or IgG 1 contrast are mixed with Sypro orange dye (Sigma), and mixture temperature is increased to 95 DEG C with 1 degree increment from 25 DEG C.In the time of temperature raising and IgG expansion, Sypro orange dye is incorporated in IgG.Monitor Sypro orange dye and IgG the produced fluorescent signal that is connected with BioRad CFX96 instrument.This analyze in, by the negative regression of Sypro orange-colored signal (negative regression) in order to identify peak value melting (that is the T of each protein _m) point.

Melt temperature (the T of Analysis deterrmination XB1511/D8 and XB2202/A4 since then _m) be respectively 67 DEG C to 70 DEG C.By this and T _migG 1 control antibodies that is 72 DEG C fully compares.This digital proof VH of the present invention and VH/VL are to being designed to the form of high heat stability total length IgG molecule.

The binding affinity of embodiment 11:XB2202VH, scFv and IgG1 antibody on human class

Use surface plasma body resonant vibration on Biacore T100, to determine XB2202VH territory, XB2202/A4scFv and the binding kinetics of XB2202/A4IgG1 to mankind PDFGR.In brief, recombinant human PDGFR-hIgG1-Fc chimeric fusion protein (R & D, #385-PR-100/CF) is fixed on the Series CM5 sensing wafer with anti-hIgG1Fc monoclonal antibody (analyzing for VH and ScFv) or anti-6His antibody (analyzing for IgG) coupling.Make XB2202VH territory, XB2202/A4scFv and XB2202/A4IgG1 flow 3 minutes and allow to dissociate 10 minutes in side from the teeth outwards with different concns (75nM, 50nM, 25nM, 10nM, 5nM and 1nM) under 50 μ l/min or 100 μ l/min.Carry out analytical data with Biacore T100 analysis software 1:1 model.Check on the quality and carry and make it avoid allowing accurate measurement.All data are all according to the dual reference in addition of Biacore standard agreement.

In table 9, show the binding kinetics of XB2202VH territory, XB2202/A4scFv and XB2202/A4IgG1 antibody on human class PDGFR β herein.Such data presentation XB2202VH territory, XB2202/A4scFv and XB2202/A4IgG1 have high binding affinity to PDGFR β separately.Especially note that the dissociation rate (2.95 × 10 with independent not paired XB2202VH territory ^-3s ^-1) compare, XB2202/A4scFv and XB2202/A4IgG1 show that improved dissociation rate (is respectively 1.54 × 10 ^-3s ^-1and 1.56 × 10 ^-3s ^-1).

Table 9:XB2202VH territory, XB2202/A4scFv and the binding kinetics of XB2202/A4IgG1 to mankind PDGFR β

Antibody	Adsorption rate (M ^-1s ^-1)	Dissociation rate (s ^-1)	Kd(M)
				XB2202VH	1.30×10 ⁷	2.95×10 ^-3	2.27×10 ^-10
XB2202/A4ScFv	7.06×10 ⁵	1.54×10 ^-3	2.18×10 ^-9
				XB2202/A4IgG1	9.80×10 ⁵	1.56×10 ^-3	1.59×10 ^-9

Embodiment 12: use in vivo mouse model to carry out the functional analysis of anti-PDGFR β antibody

Use the people such as Nobuo, Am.J.Path, (2006) 168 (6), described in 2036-2052 (mode of quoting is in full incorporated herein), assess at developing retinal vessel structural models, cornea neovascularity generation model and/or choroid neovascularity generation model the ability that anti-PDGFR β antibody disclosed herein in vivo suppresses the vascularization of PDGF inducibility.In such analysis, with the form of VH territory, scFv and/or total length IgG to mouse administration of antibodies.

Claims

Specific binding in PDGFR β through a separation and combination polypeptide, it comprises the CDR3 sequence shown in SEQ ID NO:1.
2. Binding peptide according to claim 1, it comprises VH territory, and described VH territory comprises the CDR3 aminoacid sequence shown in SEQ ID NO:1.
3. Binding peptide according to claim 2, wherein said VH territory further comprises CDR2, and described CDR2 comprises the aminoacid sequence that is selected from SEQ ID NO:2-32.
4. Binding peptide according to claim 3, wherein said VH territory further comprises CDR1, and described CDR1 comprises the aminoacid sequence that is selected from SEQ ID NO:33-62.
5. Binding peptide according to claim 4, it comprises the VH domain amino acid sequence of enjoying at least 80% amino acid sequence identity with the VH domain amino acid sequence that is selected from SEQ ID NO:318-368.
6. Binding peptide according to claim 4, wherein said VH territory comprises the aminoacid sequence that is selected from SEQ ID NO:318-368.
7. according to Binding peptide in any one of the preceding claims wherein, it comprises VL territory, and described VL territory comprises CDR3, and described CDR3 comprises the aminoacid sequence that is selected from SEQ ID NO:63-147.
8. Binding peptide according to claim 6, wherein said VL territory further comprises CDR2, and described CDR2 comprises the aminoacid sequence that is selected from SEQ ID NO:148-232.
9. Binding peptide according to claim 6, wherein said VL territory further comprises CDR1, and described CDR1 comprises the aminoacid sequence that is selected from SEQ ID NO:233-317.
10. Binding peptide according to claim 7, it comprises the VL domain amino acid sequence of enjoying at least 80% amino acid sequence identity with the VL domain amino acid sequence that is selected from SEQ ID NO:369-453.
11. Binding peptides according to claim 7, wherein said VL territory comprises the aminoacid sequence that is selected from SEQ ID NO:369-453.
12. 1 kinds of Binding peptides, it is incorporated into the identical epi-position on PDGFR β with the Binding peptide that comprises the VH domain amino acid sequence shown in SEQ ID No.318.
13. 1 kinds of Binding peptides, it is incorporated into PDGFR β with the Binding peptide competition that comprises the VH domain amino acid sequence shown in SEQ ID No.318.
14. according to the Binding peptide described in claim 12 or 13, and it comprises VH domain amino acid sequence, and described VH domain amino acid sequence is enjoyed at least 80% amino acid sequence identity with the VH domain amino acid sequence that is selected from SEQ ID NO:318-368.
15. according to Binding peptide in any one of the preceding claims wherein, and it suppresses the activity of PDGFR β.
16. Binding peptides according to claim 15, wherein the activity of PDGFR β suppresses by the combination of antagonism PDGF and PDGFR β.
17. Binding peptides according to claim 15, wherein the activity of PDGFR β suppresses by antagonism PDGFR β dimerization.
18. according to Binding peptide in any one of the preceding claims wherein, and it is incorporated into PDGFR β to be less than the Kd of 250pM.
19. according to Binding peptide in any one of the preceding claims wherein, and it is incorporated into PDGFR β to be less than the Kd of 100pM.
20. according to Binding peptide in any one of the preceding claims wherein, and it is to be less than 10 ^-3s ^-1dissociation rate be incorporated into PDGFR β.
21. according to Binding peptide in any one of the preceding claims wherein, and its specific binding is in mouse and mankind PDGFR β.
22. according to Binding peptide in any one of the preceding claims wherein, and it is to be less than the combination of IC50 antagonism PDGF and described PDGFR β of 5nM.
23. according to Binding peptide in any one of the preceding claims wherein, and it suppresses the part inducibility tyrosine phosphorylation of PDGFR β to be less than the IC50 of 4nM.
24. according to Binding peptide in any one of the preceding claims wherein, and it suppresses the migration of retina pericyte to be less than the IC50 of 6nM.
25. according to Binding peptide in any one of the preceding claims wherein, and it comprises the VH territory that melt temperature (Tm) is at least 68 DEG C.
26. according to Binding peptide in any one of the preceding claims wherein, and it is antibody.
27. according to Binding peptide in any one of the preceding claims wherein, and it is scFv.
28. 1 kinds through isolating nucleic acid, and it is encoded according to Binding peptide in any one of the preceding claims wherein.
29. 1 kinds of recombinant expression vectors, it comprises nucleic acid according to claim 28.
30. 1 kinds of host cells, it comprises recombinant expression vector according to claim 29.
31. 1 kinds produce specific binding in the method for the Binding peptide of mankind PDGFR β, and it comprises cultivates host cell according to claim 30 making described host cell produce specific binding under the condition of the Binding peptide of mankind PDGFR β.
32. 1 kinds of pharmaceutical compositions, it comprises according to the Binding peptide described in any one in claim 1 to 27 and one or more of pharmaceutically acceptable carrier.
33. 1 kinds are used for the treatment of the method for disease or illness PDGFR ss related diseases or illness, and described method comprises to the experimenter who has needs uses pharmaceutical composition according to claim 32.
34. methods according to claim 33, wherein said disease or illness are relevant macular degeneration of age (AMD) or cancer.
Various library in 35. 1 kinds of not paired VH territories, each member in wherein said library is incorporated into mankind PDGFR β.
36. libraries according to claim 35, wherein diversity is in FR1-FR3 district, and each member in wherein said library comprises the CDR3 aminoacid sequence shown in SEQ ID NO:1.
37. 1 kinds of various libraries that stable VH/VL is right, each member in wherein said library is incorporated into mankind PDGFR β.
38. according to the library described in claim 37, and each member in wherein said library comprises VH territory, and described VH territory comprises the CDR3 aminoacid sequence shown in SEQ ID NO:1.
39. according to the library described in claim 38, and wherein said VL territory is mankind VL territory.
40. according to the library described in any one in claim 35 to 39, and wherein said library is nucleic acid display libraries.
41. according to the library described in claim 40, and wherein said nucleic acid display libraries is DNA display libraries.